Binding members with altered diversity scaffold domains

ABSTRACT

This invention relates to libraries of binding members that each comprise a fusion protein which contains a donor diversity scaffold domain, such as a cysteine rich protein, inserted within a recipient diversity scaffold domain, such as an antibody constant or variable domain. Libraries and methods of generating libraries are provided, along with screening methods, binding members and methods of using the binding members.

FIELD

This invention relates to binding members with altered diversity scaffold domains, the production of libraries of such binding members and the selection and screening of binding members from such libraries.

BACKGROUND

Antibody humanisation involves combining animal CDRs with human frameworks. CDR grafting is a well-established method used for antibody humanisation. Antibodies generated by immunisation of an animal are sequenced, CDR loops identified and a hybrid molecule is prepared consisting of a human framework with one or more CDRs derived for the animal antibody¹. In CDR grafting, it is likely that the incoming peptide is required to adopt a particular structure to maintain the original binding specificity and it is often necessary to “fine-tune” the humanisation process by changing addition residues including supporting residues within the framework region.

Non-antibody peptides from other sources have also been transplanted into antibody frameworks. Barbas et al (1993) inserted a naturally-occurring integrin binding sequence in to the VH CDR3 region of a human antibody² and Frederickson et al (2006)³ cloned a 14-mer peptide which was known to bind to the thrombopoietin (TPO) receptor into the several CDR regions of an anti-tetanus toxoid antibody. 2 amino acids at each end of the peptide were randomized and the resultant library selected by phage display. This same work was described in US 20100041012A1, along with insertion of an 18 amino acid peptide known to bind to the erythropoietin receptor⁴. The original erythropoietin receptor binding peptide sequence contained two cysteines which were removed due to anticipated problems with antibody disulphide bond formation. In these cases, 2-3 codons were randomised at the junction between the inserted peptide coding region and the recipient framework to select optimal junctional sequences from the resultant phage display libraries. Liu et al inserted a 14-16 amino acid peptide with binding activity to CXCR4 into 2 different CDRs (VH CDR3 and VH CDR2⁵). Kogelberg inserted an αVβ6 17 mer peptide which adopts a hairpin:alpha helix motif, into VH CDR3 of a murine antibody⁶. This peptide contained core RGDLxxL element known to direct binding to αVβ6. This work led on to a library-based approach focussed on VH CDR3, which sought to mimic the hairpin:alpha helix structure⁷ of the original insert. In all these cases, the antibody acted as a carrier for the incoming peptide without contributing to target binding.

Gallo et al have inserted peptides into a VH CDR3 using an alternative approach for diversification. Antibody diversification naturally occurs within in B cells and U.S. Pat. No. 8,012,714 describes a method replicating this diversification process within heterologous mammalian cells. In maturing B cells antibody genes are assembled by recombination of separate V, D and J segments through the activities of “recombination activating genes” (RAG1 and RAG2) resulting in deletion of large segments of intervening sequences. In U.S. Pat. No. 8,012,714, RAG1, and RAG2 were introduced into human embryonic kidney cells (HEK293) along with plasmids encompassing recombination substrates to replicate recombination V-D-J joining in non-B cells. This RAG1/2 recombination-based approach utilizes DNA repair and excision mechanisms of mammalian cells and has not been demonstrated in prokaryotic cells.

The same mammalian cell-based in vitro system was used in WO2013134881A1 with the D segment being effectively replaced by DNA which encodes peptide fragments derived from two other binding molecules. Since RAG1/RAG2 driven diversification only occurs in mammalian cells this system cannot be used directly in prokaryotic approaches such as phage display. WO2013134881A1 exemplifies the introduction into the heavy chain CDR3 of the same 14-mer peptide fragments used by Frederickson³ and Bowdish (US20100041012 A1). The TPO receptor binding construct was introduced into the middle of CDR3 and so retained the CDR sequences as well as grafting in additional amino acids to create a total of 12 or more amino acids between the antibody framework and the incoming peptide. WO2013134881A1 also exemplifies antibody fusions with extendin-4 which were generated with combined linker length between the antibody framework and the incoming peptide ranging from 9-14 amino acids.

Saini et al^(8, 9) reported the occurrence of ultra-long cysteine-rich CDR3s within the VH domain of bovine antibodies. These ultra-long VH CDRs of 40-67 amino acids which constitute 9% of the bovine antibody response were typically restricted to pairing with a specific Vλ1 light chains. Recently the structure of two such bovine antibodies has been determined through X ray crystallography¹⁰ revealing an unusual stalk structure formed by an ascending and descending β strands which is topped by a “knob” comprising a peptide with multiple disulphide bonds formed from cysteine pairs. This stalk: knob structure is formed by CDR3 of the VH domain but the stalk is supported by interactions with VH CDR1, VH CDR2, as well as all CDRs of the partner Vλ1 chain. Thus the entire CDR diversity of these antibodies is devoted to the presentation of the “knob” structure which itself is presented at a distance of approximately 38 Angstroms from the supporting framework to the first cysteine of the “knob”¹¹. The potential of the other CDRs to interact with the same epitope recognized by the knob is therefore abolished or severely limited.

In subsequent work, “the knob” was replaced by cytokines, such as granulocyte colony stimulating factor¹² and erythropoietin¹³, which were linked to the stalk by a flexible Gly-Gly-Gly-Gly-Ser (SEQ ID NO: 1) sequence (Gly4Ser) at the N and C terminal junctions. It was subsequently shown that the rigid β sheet stalk structure could be replaced with a rigid anti-parallel coiled coil structure¹¹. Flexible Gly4Ser and Gly-Gly-Ser-Gly (SEQ ID NO: 2) linkers were again placed at each end of the coiled: coil sequence to “optimize the folding and stability of the resulting antibody”. This construction also allowed the presentation granulocyte colony stimulating factor. Liu et al have taken the same approach of using rigid stalk structures coupled with a flexible peptides at both junctions to fuse leptin and Growth Hormone into VH CDR3 and VH CDR2 and VL CDR3¹⁴. In this example, retained antibody binding was seen as a potential disadvantage and the suggestion was made of using a “null” antibody partner e.g. anti-RSV antibody to avoid contribution of binding of the host antibody.

Peng et al (2015) reports the insertion of two natural agonists (leptin and follicle stimulating hormone) as “guests” into an antibody VH CDR3 in a way which retained agonistic activity¹⁵. A library of fusions was created wherein flexible peptides of 5-15 amino acids were inserted between the domains at each of the N terminal and C terminal ends along with a random 3 amino acids at each end (i.e. total linker lengths of 16-36 amino acids). A library of 10⁷ fusion variants was expressed in HEK293 cells and fusions which retained agonist activity of the “guest” were identified through reporter gene activation in an autocrine-based screening approach. The beneficial half-life properties of the antibody are extended to the incoming guest.

The approach of Peng et al (2015) was carried out in mammalian cells using an autocrine reporter system wherein cells are grown in semi-solid medium and secreted antibody fusions are retained in the vicinity of the producing cell. Thus, relatively high concentrations of the secreted product accumulated in the immediate vicinity of the cell¹⁶ making it difficult to distinguish clones within a library with altered expression/stability or binding properties. Thus, there is a limitation on stringency within the system described by Peng et al (2015). Furthermore the functional screening approach is also limited to screens for which a relevant receptor/reporter system is available. The proportion of clones which worked was low (despite the benefit of endogenous chaperone-assisted folding within mammalian cells).

Antibodies represent a commonly used binding scaffold. As an alternative, the generation of binding members based on non-antibody scaffolds has been demonstrated^(38, 39). These engineered proteins are derived from libraries of variants wherein exposed surface residues are diversified upon a core scaffold³⁸⁻⁴⁰.

Betton et al (1997)¹⁷ reports fusing β lactamase into several accommodating permissive sites in maltose binding proteins using 9-10 amino acid linkers (DPGG-insert-YPGDP (SEQ ID NOS: 3, 4) and PDPGG-insert-YPGGP (SEQ ID NOS: 5, 6). Collinet¹⁸ reports inserting P lactamase or dihydrofolate reductase into permissive sites of phosphoglycerate kinase using amino acid residues SGG or GG at the N terminal insertion point and GAG at the C terminal insertion point. Studies of the enzyme β-lactamase, which confers bacterial resistance to ampicillin, have also revealed potential sites into which peptide insertions could be accommodated¹⁹. Vandevenne et al reports the insertion into one of these sites of a chitin binding domain protein of the human macrophage chitotriosidase²⁰, using a construct that introduced 6-7 amino acids each at the N and C termini of the insertion. Crasson et al²¹ have demonstrated functionalization of a nanobody by inserting it into a permissive site of β lactamase joining two helices.

SUMMARY

The present inventors have found that the incorporation of an entire binding domain (“donor diversity scaffold domain”) within a second binding domain (“a recipient scaffold domain”) increases the diversity of populations of binding members and allows the selection and isolation of binding members with advantageous properties from these populations. This may be useful for example in selecting binding members which bind to target molecules, such as ion channels, that are difficult to target with conventional antibodies.

An aspect of the invention provides a method of screening comprising;

-   -   (i) providing a founder library of binding members,     -   each binding member in the founder library comprising a fusion         protein and optionally, a partner domain associated with the         fusion protein,     -   wherein the fusion protein comprises a donor diversity scaffold         domain inserted within a recipient diversity scaffold domain,         optionally wherein the N and C terminals of the donor diversity         scaffold domain are each linked to the recipient diversity         scaffold domain with linkers of 4 amino acids or fewer each,     -   wherein the donor diversity scaffold domain comprises a donor         scaffold and a donor interaction sequence and the recipient         diversity scaffold domain comprises a recipient scaffold and a         recipient interaction sequence, and one or more of the donor         interaction sequence, recipient interaction sequence and the         linkers are diverse in said founder library,     -   (ii) screening the founder library for founder binding members         which display binding activity, and     -   (iii) identifying one or more founder binding members in the         founder library which display the binding activity.

In some embodiments, the linkers are diverse in said founder library.

The method may further comprise;

-   -   (iv) introducing diverse amino acid residues at one or more         positions in the amino acid sequence of one or more identified         founder binding members to produce a modified library of binding         members,     -   (v) screening the modified library for modified binding members         which display a binding activity and     -   (vi) identifying one or more modified binding members in the         modified library which display the binding activity.

In some embodiments, diverse amino acid residues may be introduced into the donor interaction sequence, recipient interaction sequence and/or partner domain of the one or more identified founder binding members.

The method may further comprise;

-   -   (vii) associating the fusion protein from the one or more         identified binding members or modified binding members with a         diverse population of partner binding domains (partner chains)         to produce a shuffled library of binding members,     -   (viii) screening the shuffled library for shuffled binding         members which display a binding activity,     -   (ix) identifying one or more shuffled binding members which         display the binding activity.

Another aspect of the invention provides a method of producing a library of binding members comprising;

-   -   providing a population of nucleic acids encoding a diverse         population of fusion proteins comprising a donor diversity         scaffold domain inserted into a recipient diversity scaffold         domain, optionally wherein the N and C terminals of the donor         diversity scaffold domain are linked to the recipient diversity         scaffold domain with linkers of 4 amino acids or fewer at each         terminal,     -   wherein the donor diversity scaffold domain comprises a donor         scaffold and a donor interaction sequence and the recipient         diversity scaffold domain comprises a recipient scaffold and a         recipient interaction sequence, and     -   one or more of the donor interaction sequence, recipient         interaction sequence and linkers are diverse in said population,     -   expressing said population of nucleic acids to produce the         diverse population, and     -   optionally associating the fusion proteins with a population of         partner domains. thereby producing a library of binding members.

Another aspect of the invention provides a library of binding members comprising;

-   -   a diverse population of fusion proteins comprising a donor         diversity scaffold domain inserted into a recipient diversity         scaffold domain,     -   wherein the donor diversity scaffold domain comprises a donor         scaffold and a donor interaction sequence and the recipient         diversity scaffold domain comprises a recipient scaffold and a         recipient interaction sequence, optionally wherein the N and C         terminals of the donor diversity scaffold domain are each linked         to the recipient diversity scaffold domain with linkers of 4         amino acids or fewer,     -   wherein the donor diversity scaffold domain comprises a donor         scaffold and a donor interaction sequence and the recipient         diversity scaffold domain comprises a recipient scaffold and a         recipient interaction sequence, and     -   one or more of the donor interaction sequence, recipient         interaction sequence and linkers are diverse in said population,         and     -   optionally wherein the fusion proteins are associated with         binding partners to form heterodimers.

Another aspect of the invention provides a fusion protein comprising a donor diversity scaffold domain inserted into a recipient diversity scaffold domain

-   -   wherein the donor diversity scaffold domain comprises a donor         scaffold and a donor interaction sequence and the recipient         diversity scaffold domain comprises a recipient scaffold and a         recipient interaction sequence, optionally wherein the N and C         terminals of the donor diversity scaffold domain are linked to         the recipient diversity scaffold domain with linkers of 4 amino         acids or fewer.

Another aspect of the invention provides a binding member comprising a fusion protein described herein and a partner domain associated with the fusion protein.

Other aspects of the invention provide isolated donor diversity scaffold domains, recipient diversity scaffold domains or partner domains from binding members identified and/or isolated as described herein, for example isolated donor diversity scaffold domains engineered as described herein or isolated recipient diversity scaffold domains or partner domains which may be useful in heterologous binding members, for example binding members which do not include the original donor diversity scaffold domain.

BRIEF DESCRIPTION OF FIGURES

FIG. 1A shows a representation of pIONTAS1, showing the cassette encompassing the lacI promoter, M13 leader sequence scFv antibody gene-gene 3 (not to scale). This cassette is present within the Hind3/EcoR1 cloning sites of pUC119 which has an Ampicillin resistance gene and a coleE1 origin and an f1 bacteriophage origin of replication. The VH gene is flanked by an Nco1 and Xho1 restriction site and the VL gene is flanked by Nhe1 and Not 1 restriction sites allowing simple cloning into compatible vectors. FIG. 1B shows a representation of the VL domain showing positions of framework regions (FW) and CDR regions. A knottin donor has been inserted at CDR1 position of the VL recipient. Pml1 and Mfe1 sites allow cloning of the knottin donor library. The region from FR2 to the end of FR4 is derived from a repertoire of light chains²⁸. FIG. 1C is as described for FIG. 1B but with the knottin donor replacing CDR2 of the VL gene using Pst1 and BspE1 sites.

FIG. 2 shows the sequence of synthetic single chain Fv genes used in KnotBody construction. The VH of an existing antibody D12 was used within KnotBody constructs. FIG. 2A shows restriction sites Nco1 and Xho1 which flank the VH sequence as well as the flexible linker joining VH to VL. This is followed by a sequence encoding the Vlamda1a (IGLV1-36) germline. CDR1 of the VL is shown and is preceded by a Pml1 cloning site. A Ser to Thr mutation was introduced to enable the inclusion of a Pml1 site at the end of framework 1. This site allows cloning of the knottin into the CDR1 position of the VL. At the end of VL framework 2 of the synthetic gene is a Pst1 site allowing cloning of a knottin into the CDR2 position. FIG. 2B is as for FIG. 2A but showing the V kappa germline sequence IGKV1D-39

FIG. 3 shows the insertion of EETI-II donor knottin into VL recipient. FIG. 3A shows the sequence of knottin donor EETI-II with cysteine residues emboldened and core sequence involved in trypsin binding (“PRIL”) underlined. FIG. 3B shows a representation of the insertion of EETI-II donor into CDR1 or CDR2 of the lambda germline gene IGLV1-36. Boundaries are defined according to the international ImMunoGeneTics information system (IMGT)²⁹. Amino acids are represented by IUPAC single letter amino acids code. FIG. 3Bi shows the sequence of the V lambda recipient domain at CDR1 before insertion of the knottin. FIGS. 3 ii and 3 iii show the sequence after insertion of the donor EETI-II donor. (FIG. 3Biii is generated by a primer which introduced an extra Gly residue between donor and framework residues compared to 3B ii). FIG. 3B iv show the sequence of the V lambda recipient domain at CDR2 before donor insertion and 3B v shows after insertion of the EETI-II donor. Antibody framework residues are shown underlined and residues from the antibody recipient which are lost are shown in lower case. At the junctions, randomising residues are shown as x with those which replace framework or donor residues shown as x in lower case. Any “additional” residues within the linker between the domains are shown emboldened in upper case (A, Z or X). Z means any amino acid from Val, Ala, Asp or Gly. X represents the amino acids encoded by the codon VNC (encoding 12 amino acids) or VNS encoding 16 amino acids in total. (V=A, C or G and S=C or G). FIGS. 3C-F show the DNA and amino acid sequence of the construct arising from insertion of the EETI-II knottin donor into respectively C. CDR1 of IGLV1-36, D. CDR1 of IGKV1D-39, E. CDR2 of IGLV1-36 and F. CDR2 of IGKV1D-39. (IGLV1-36 is a V lambda germline gene and IGKV1D-39 is a V kappa germline gene). Restriction sites used in cloning are highlighted and the framework and CDR regions are identified.

FIG. 4 shows an analysis of polyclonal phage from round-2 selection output. The polyclonal phage (X-axis) from round 2 panning of EETI-II CDR1 library (B) and EETI-II CDR2 library (C) were tested for binding to trypsin. Trypsin binding was not achieved when EETI-II directly fused to the N-terminus of gene-III (A). The Y-axis shows trypsin binding in fluorescent units (FU).

FIG. 5 shows a monoclonal binding assay of KnotBody clones from round-2 selection output. 94 individual clones from EETI-II CDR1 (A) and EETI-II CDR2 (B) library selection output (round-2) were picked into 96 well culture plates and phage were prepared from each clone. The phage supernatant from each clone was tested for binding to biotinylated trypsin immobilised on neutravidin coated Maxisorp™ plates. Phage binding to trypsin was detected using a mouse anti-M13 antibody followed by europium conjugated anti-mouse antibody. The X-axis shows the clone number and the Y-axis shows trypsin binding in fluorescent units (FU).

FIG. 6 shows western blot analysis of KnotBody clones and their GlyAla mutants. Phage prepared from 11 (randomly chosen) KnotBodies (referred to as W1-W11) and their GlyAla mutants (referred to as M1-M11) were analysed on a western blot using mouse anti-pIII antibody. Wild type (WT) gene III and KnotBody (KB)-gene III fusion were visualised by an anti-mouse IRDye® 680 (LI-COR, Cat. No. 926-32220) secondary antibody.

FIG. 7 shows KnotBody Fab expression cassette in pINT12 vector system. In this plasmid, transcription of light chain cassette encoding the EETI-II VL fusion and CL domain is under the control of EF1-alpha promoter. Transcription of the heavy chain cassette encoding D1A12 VH fused to IgG1 constant domain 1(CH1) is controlled by the CMV promoter.

FIG. 8A shows SDS-PAGE analysis of IgG1 KnotBodies expressed in HEK-293F cells (representative example). KnotBody Fabs purified using CaptureSelect affinity matrix were visualised on a SDS-PAGE gel using Coomassie staining. A major band of approximately 50 kD observed for samples prepared under non-reducing conditions correspond to the complete KnotBody Fab molecule (Lane 2 and 4). The upper band (approximately 27 kD) and the lower band (approximately 23 kD) observed for samples prepared under reducing conditions (Lane 3 and 5) correspond to VH-CH1 fusion and Knottin-VL-CL fusion respectively. Lane 1 is a loaded with Biorad protein ladder (Biorad, 161-0373). FIG. 8B shows the chromatographic profile of a KnotBody analysed by size exclusion chromatography using over a Superdex200 PC3.2/30 column (GE Healthcare).

FIG. 9 shows the structure of EETI-Antibody VL CDR2 fusion. Crystal structure of KB_A05 (A) at 1.9 Å and crystal structure of KB_A12 (B) at 2.5 Å.

FIG. 10A (i) shows previously published structure of EETI-II shown in ribbon mode (PDB code 1H9I). FIG. 10A (ii) EETI-II as VL CDR2 fusion. FIG. 10A (iii) Superimposition of previously published EETI-II structure and EETI-II as VL CDR2 fusion. FIG. 10B shows VH and VL (fused to EETI-II) of Fab KB_A05. VH CDR residues are highlighted in black and VH CDR3 is annotated.

FIG. 11 shows an analysis of polyclonal phage outputs from the phage display selection of KnotBody VH shuffled libraries. Heavy chain shuffled KB_A07 and KB_A12 (combined) libraries were selected on 5 different antigens: cMET-Fc (A), GAS6-CD4 (B), FGFR4-CD4 (C), uPA (D) and B-gal (E). The polyclonal phage from the 5 selections were each tested for binding to trypsin, all the antigens used in selection, neutravidin and streptavidin to determine background binding. X-axis shows antigens immobilised on Maxisorp™ plates and the Y-axis shows polyclonal phage binding to the antigen in fluorescent units (FU).

FIG. 12 shows representative examples of monoclonal bi-specific KnotBodies (from cMET-Fc and GAS6-CD4 selections) binding to trypsin and cMET-Fc or trypsin and GAS6-CD4. Monoclonal phage binding to the antigens immobilised on Maxisorp™ plates were detected using a mouse anti-M13 antibody followed by europium conjugated anti-mouse antibody. X-axis shows the clone number and the Y-axis shows antigen binding in fluorescent units (FU).

FIG. 13 shows phage selection cascade from the ‘heavy chain shuffled’ KnotBody library selected on biotinylated trypsin. The optimum antigen concentrations for round-2 and round-3 were determined empirically by selecting the phage KnotBodies against range of trypsin concentrations and comparing the output numbers with a “no antigen control”.

FIG. 14 shows the format of the KnotBody capture assay. KnotBody-scFv-Fcs (B) were captured on Maxisorp™ plates coated with an anti-Fc antibody (A) and the binding biotinylated trypsin (C) to KnotBodies are detected using streptavidin conjugated europium (D).

FIG. 15 shows a representative example of the performance of affinity improved clones in KnotBody Fc capture assay. KnotBody-scFv-Fcs were captured on Maxisorp™ plates coated with an anti-Fc antibody and the binding biotinylated trypsin to KnotBodies are detected using streptavidin conjugated europium. The binding signals observed for clones isolated from affinity maturation selections were compared to the parent clones KB_A12 and KB_A07. The X-axis shows clone ID of KnotBodies and the Y-axis shows KnotBody binding to trypsin in fluorescent units.

FIG. 16 shows an improvement in trypsin binding after heavy chain shuffling. Off-rate of the clone KB_Tr_F03 selected from the heavy chain shuffled library is compared to the two parent KnotBodies KB_A07 and KB_A12. The X-axis shows off rate analysis in seconds and the Y-axis shows resonance units (RU).

FIG. 17 shows a KnotBody expression cassette in pINT3-hg1 vector system. In this plasmid, transcription of light chain cassette encoding the knottin/toxin VL fusion and CL domain is under the control of EF1-alpha promoter. Transcription of the heavy chain cassette encoding D1A12 VH and IgG1 constant domains (CH1-CH2-CH3) is controlled by the CMV promoter. Pst1 and BspE1 sites in the light chain gene (recipient) allow the insertion of knottins (donor) into the CDR2 position.

FIG. 18 shows SDS-PAGE analysis of IgG1 KnotBodies expressed in HEK-293F cells. KnotBodies purified using Protein-A affinity chromatography were visualised on a reducing SDS-PAGE gel using Coomassie staining. The top bands correspond to the antibody heavy chains (HC) and the bottom bands corresponds the antibody light chain displaying knottins/toxins as CDR2 fusions. Samples 1-4 are KB_A12 antibody recipient fused NaV1.7 blockers (Huwentoxin-IV, ProTx-II, Ssm6a, and Ssm6a_GGS). Samples 5-10 are Kv1.3 blockers (Kaliotoxin, Moka-1 and Shk) fused to KB_A07 antibody (5, 6 and 7) or KB_A12 antibody (8, 9 and 10). Samples 11 and 12 are parental KnotBody clones (EETI-II fusions) KB_A07 and KB_A12 respectively.

FIG. 19 show specific binding of two adhiron-antibody fusions to LOX-1 protein. In this assay, binding of phage displaying adhiron-antibody fusions to LOX1 was detected using a mouse anti-M13 antibody followed by europium conjugated anti-mouse antibody. Parent KnotBodies KB_A07 and KB_A12 (EETI-II fusions) were included as controls. Proteins cMET-FC, GAS6-CD4, FGFR4-CD4, UPA and B-gal were used to examine non-specific binding. All antigens were directly immobilised on Maxisorp™ plates.

FIG. 20 shows a representation secondary structure overlaid on primary sequence FIG. 20A shows the sequence of 10^(th) type III cell adhesion domain of fibronectin. Secondary structural elements (beta sheets) are underlined. The residues which join the beta sheets on the upper face of the domain are shown lower case. FIG. 20B shows the sequence of gp2 protein with beta sheets and alpha helical regions labelled. Potential sites for donor insertion or diversification are shown in lower case. N and C terminal residues removed without affecting structure are shown in italics and smaller font.

FIG. 21 represents a concentration-response curves showing the concentration dependent inhibition of huKv1.3 channel currents by KnotBodies. Log [M] concentration (x-axis) of toxin fusion KnotBodies KB_A12_Shk (Shk toxin, squares), KB_A07_Shk (Shk toxin, triangles) and KB_A07_KTX (kaliotoxin, circles) plotted versus % current remaining (y-axis). From these concentration-response curves the concentration at which 50% current inhibition (IC₅₀; see Table 11 for summary) was determined.

FIG. 22 shows the binding comparison of KnotBody linker variants to trypsin. Binding of KnotBody Fabs (X-axis) to biotinylated trypsin immobilised on streptavidin coated Maxisorp™ plates using a mouse anti-CH1 antibody followed by a europium conjugated anti-mouse antibody. Y-axis shows normalised binding signal (%) where the binding of Gly4Ser controls are normalised against that of their respective parent clone (i.e. 100% binding).

FIG. 23 shows the monoclonal phage binding of clones selected from the KnotBody loop library to β-galactosidase (A) and cMET (B). Monoclonal phage binding to the antigen immobilised directly on Maxisorp™ plates were detected using a mouse anti-M13 antibody followed by europium conjugated anti-mouse antibody. X-axis shows the clone number and the Y-axis shows antigen binding in fluorescent units (FU).

FIG. 24 shows a representative example of the screen for KnotBody binders to rat TFR. Monoclonal phage binding to rat TFR immobilised on Maxisorp™ plates were detected using a mouse anti-M13 antibody followed by europium conjugated anti-mouse antibody. X-axis shows the clone number and the Y-axis shows signal from phage binding to TFR in fluorescent units (FU).

FIG. 25 shows the binding comparison of KnotBody KB_A12 and a format consisting of a bovine antibody with a natural “ultra long VH CDR3 (“cow ULVC-Ab”). The cysteine rich knob of the cow ULVC-Ab was replaced by the EETI-II knottin (“EETI-II cow ULVC-Ab”). Genes encoding these constructs were cloned into pSANG4 and phage rescued from either KB_A12 or the EETI-II cow ULVC-Ab fusion constructs (X-axis) and was PEG precipitated. 12.5× (A) and 0.5× (B) phage (relative to the initial culture volume) were tested for binding against biotinylated trypsin immobilised on streptavidin coated Maxisorp™ plates. Phage binding to trypsin was detected using an anti-M13 antibody followed by europium conjugated anti-mouse antibody. Y axis shows the phage binding signal in fluorescent units (FU).

FIG. 26 shows a schematic representation of the mammalian display vector pD6. Antibody genes are targeted to the human AAVS locus which is located within a 4428 bp intron between the first and second exons of the gene encoding protein phosphatase 1, regulatory subunit 12C, PPP1R12C. A pair of Tale nucleases directed to this region are used to cleave the genome at this site. 700-800 bp of the sequences on the 5′ and 3′ site of the cleavage site are incorporated into donor vectors as left and right homology arms (HA) respectively. The pD6 donor vector is used to insert antibody genes formatted as single chain Fv (scFv) fused with the Fc region of human IgG1. The vector also has a CMV promoter driving expression of the scFv-Fc gene and an exon encoding the transmembrane domain from the “Platelet derived growth factor receptor (PDGFR-TM). The transgene region encoding the scFv-Fc antibody fusion is flanked by left and right homology arms (left HA, right HA) representing the sequences which flank the cleavage site in the AAVS locus. The vector also encodes a promoter-less blasticidin gene which becomes activated by in-frame splicing to exon 1 within the AAVS locus.

FIG. 27 shows the functional expression of KnotBodies on the cell surface. All cells were stained with PE labelled anti-Fc (FL2 channel) and APC labelled streptavidin/biotinylated trypsin complex (FL4 channel). Following transfection cells were selected in blasticidin and analysed by flow cytometry at 15 days post transfection. Panels show the results for (A) KB_A07 (B) KB_A12 or (C) untransfected HEK293 cells.

FIG. 28 shows a representation of the insertion of EETI-II donor knottin into VH CDR1 (A), VH CDR2 (B), VH CDR3 (C), VL CDR1 (D), VL CDR2 (E) and VL CDR3 (F) of the anti-TACE antibody D1A12 scFv (Table 23, Tape, C. J., PNAS USA 108, 5578-5583 (2011)). In addition, a linker library was created for KB_A07 where the EETI-II donor knottin was inserted into VL CDR2 (G). Boundaries in this example were defined according to the V BASE database (MRC, Cambridge UK) according to criteria defined by Chothia (Chothia et al, J. Mol. Biol. 264, 220-232 (1996)). Amino acids are represented by IUPAC single letter amino acids code. FIG. 28Ai shows the sequence of the VH recipient domain at CDR1 before insertion of the knottin. FIG. 28Aii shows the sequence after insertion of the donor EETI-II donor. FIG. 28Bi shows the sequence of the VH recipient domain at CDR2 before insertion of the knottin. FIG. 28Bii shows the sequence after insertion of the donor EETI-II donor. FIG. 28Ci shows the sequence of the VH recipient domain at CDR3 before insertion of the knottin. FIG. 28Cii shows the sequence after insertion of the donor EETI-II donor. FIG. 28Di shows the sequence of the VL recipient domain at CDR1 before insertion of the knottin. FIG. 28Dii shows the sequence after insertion of the donor EETI-II donor. FIG. 28Ei shows the sequence of the VL recipient domain at CDR2 before insertion of the knottin. FIG. 28Eii shows the sequence after insertion of the donor EETI-II donor. FIG. 28Fi shows the sequence of the VL recipient domain at CDR3 before insertion of the knottin. FIG. 28Fii shows the sequence after insertion of the donor EETI-II donor. FIG. 28Gi shows the sequence of the “wild-type” KB_A07 VL recipient domain at CDR2 before linker randomisation. FIG. 28Gii shows the sequence after randomization of the linker residues. At the junctions, randomising residues are shown as X.

FIG. 29 shows the insertion of the alternatively framed 5-3 EETI-II knottin donor into VL recipient. Alternatively framed knottins were inserted into CDR1 or CDR2 of a V kappa (IGKV1 D-39) or V lambda (IGLV1-36) light chain. FIG. 29A shows the nucleic acid and amino acid sequence of the alternatively framed knottin construct EET 5-3 which is used as a donor into CDR1 and CDR2 of the VL recipient. Cysteine residues are emboldened and core sequence involved in trypsin binding (“PRIL”) underlined. FIG. 29B shows a representation of the insertion of 5-3 EETI-II donor into CDR1 or CDR2 of the lambda germline gene IGLV1-36. Boundaries are defined according to the international ImMunoGeneTics information system (IMGT)²⁹. Amino acids are represented by IUPAC single letter amino acids code. FIG. 29Bi shows the sequence of the V lambda recipient domain at CDR1 before insertion of the knottin. FIGS. 29Bii and 29Biii show the sequence after insertion of the 5-3 EETI-II donor. (The sequence in FIG. 29Biii is generated by a primer which introduced an extra Gly residue between donor and framework residues compared to 29B ii). FIG. 29B iv show the sequence of the V lambda recipient domain at CDR2 before donor insertion and 29B v shows after insertion of the EETI-II donor. Antibody framework residues are shown underlined and residues from the antibody recipient which are lost are shown in lower case. At the junctions, randomising residues are shown as x with those which replace framework or donor residues shown as x in lower case. Any “additional” residues within the linker between the domains are shown emboldened in upper case (A, Z or X). Z means any amino acid from Val, Ala, Asp, Gly or Glu (encoded by GNS codon). X represents the amino acids encoded by the codon VNC (encoding 12 amino acids) or VNS encoding 16 amino acids in total. (V=A, C or G and S=C or G). FIGS. 29 C-D show the DNA and amino acid sequence of the construct arising from insertion of the 5-3 EETI-II knottin donor into either CDR1 (C) or CDR2 of IGLV1-36 (D). The same 5-3 EET format was also introduced into CDR1 and CDR2 of IGKV1 D-39 which is a V kappa germline gene and the depiction of this is shown in FIG. 3 using the native (1-5) orientation of EETI-II, but applying the same approach as described in example 16 and depicted in FIG. 29 for insertion of the 5-3 EETI-II format. Restriction sites used in cloning are highlighted and the framework and CDR regions are identified.

FIG. 30 shows that a KnotBody comprising a Kv1.3 blocking donor domain (Shk) reduces cytokine secretion in T cells. PBMCs were activated with an anti-CD3 antibody and were co-incubated with a KnotBody in which the donor diversity domain is either Shk (KB_A12_Shk) or EET (KB_A12). Free Shk is also used as a positive control. Graphs show the levels of Interferon gamma (IFNγ) (FIG. 30A); Granzyme (FIG. 30B), Interleukin 7A (IL17A) (FIG. 30C), and TNF-alpha (FIG. 30D) following T cell activation in PBMCs.

DETAILED DESCRIPTION

This invention relates to diverse populations and libraries of binding members that comprise a donor diversity scaffold domain inserted within a recipient diversity scaffold domain, and binding members isolated from such populations and libraries.

The present inventors have shown that an entire donor diversity scaffold domain is capable of folding correctly into a stable functional conformation following insertion into a recipient binding domain, with both domains remaining biologically active. The recipient binding domain is found to accommodate the incoming donor domain without its structure being compromised. Furthermore, donor and recipient domains with multiple cysteine residues each form the correct disulphide bond patterns within the fusion protein.

Combinations of donor and recipient diversity scaffold domains as described herein are useful in increasing the diversity of the populations and libraries of binding members. For example, antibodies are naturally-evolved diversity scaffolds but most of the diversity is focussed within the CDR3 region and in particular the CDR3 region of the antibody heavy chain. The introduction of a donor diversity scaffold domain into a recipient antibody VH and/or VL domain greatly increases the available diversity of the chimeric donor/antibody binding member.

Furthermore, if the donor diversity scaffold domain is predisposed to binding a particular class of target molecule, such as an ion channel, this predisposition may be conferred on the binding member comprising the donor diversity scaffold domain. Thus, the binding member may retain the binding activity of the donor diversity scaffold domain (for example, knottin binding to an ion channel) whilst also displaying the in vivo half-life of the recipient diversity scaffold domain (for example, an antibody).

Preferably, the donor and recipient diversity scaffold domains of the chimeric binding member of the invention are joined directly together or linked by short linkers to limit or constrain flexibility and relative movement between the domains, so that the binding member is relatively rigid. Longer, unstructured linkers are more liable to proteolytic degradation and may also facilitate the formation of incorrect disulphide bonds through increased conformational flexibility. In addition, the conformational flexibility of longer, relatively unstructured peptides compromises the affinity of interaction. In an interaction between 2 molecules, such as an antibody and an antigen, conformational entropy is lost upon complex formation. With longer linkers between donor and recipient diversity scaffold domains, there are potentially many more conformations available, making complex formation energetically unfavourable. In contrast, complex formation involving a more structured fusion protein with short or absent linkers will be entropically more favourable, potentially leading to a higher affinity interaction^(30,31,32,33).

Since conformational flexibility carries a thermodynamic “entropic penalty” for binding interactions, constraining the conformational relationship between the donor and recipient diversity scaffold domains as described herein minimises the entropic penalty of interactions, with a resultant benefit in affinity of the resulting interaction of the binding member.

Given the close proximity of the donor and recipient diversity scaffold domains, amino acids on the surface of both donor and recipient diversity scaffold domains may contribute to the paratope that binds to the target molecule or different target molecules within a complex. This may increase affinity or specificity of the binding member compared to either diversity scaffold domain alone. A partner domain of either the donor or recipient diversity scaffold domain may also contribute to binding of target molecule. Any of the donor, recipient or partner domains may contact closely apposed sites on a target molecule or complex.

A binding member described herein may comprise a fusion protein. The fusion protein is a chimeric molecule comprising two diversity scaffold domains; a donor diversity scaffold domain and a recipient diversity scaffold domain.

The donor diversity scaffold domain is located within the recipient diversity scaffold domain i.e. the donor diversity scaffold domain is flanked at both its N and C termini by the recipient diversity scaffold domain.

Preferably, both the donor and recipient diversity scaffold domains of the fusion protein contribute to the binding activity of the fusion protein, for example binding to the same or different target molecules.

In other embodiments, one of the donor and recipient diversity scaffold domains of the fusion protein, preferably the donor diversity scaffold domain is responsible for the binding activity of the fusion protein, for example the binding of the target molecule.

In some preferred embodiments, the fusion protein is suitable for display on the surface of a bacteriophage, for example for selection by phage display. A fusion protein for display may further comprise a phage coat protein, such as pIII, pVI, pVIII, pVII and pIX from Ff phage or the gene 10 capsid protein of T7 phage. The phage coat protein may be located at the N or the C terminal of the fusion protein, preferably at the C terminal of the fusion protein.

A diversity scaffold domain is an independently folding structural domain with a stable tertiary structure which is able to present diverse interaction sequences with potential to mediate binding to a target molecule. Diversity scaffold domains include domains represented within paralogous or orthologous groups which have been utilized in evolution for driving interactions of said scaffold with other molecules. Diversity scaffold domains also include natural domains which have been engineered to display diversity as well as entirely synthetic proteins evolved or designed to form a stable self-folding structure.

Examples of diversity scaffold domains known in the art include immunoglobulin domains⁴¹, cysteine-rich peptides, such as knottins and venom toxin peptides, affibodies (engineered Z domain of Protein A domain)^(42, 43), monobodies (i.e. engineered fibronectins)⁴⁴, designed ankyrin repeat proteins (DARPins)⁴⁵, adhirons⁴⁶, anticalins⁴⁷, thioredoxin⁴⁸, single domain antibodies^(49, 50) and T7 phage gene 2 protein (Gp2)⁵¹.

In some preferred embodiments, a diversity scaffold domain may comprise multiple disulphide bridges formed by cysteine residues within the scaffold domain.

Preferred diversity scaffold domains for use as described herein include the VH and VL domains of an antibody. The binding of T cell receptors to their targets is also directed by CDR loops present within variable Ig domains (a and (3) with greatest diversity present in CDR3 of the β chain. These have been used in vitro to present diverse sequences^(34, 35). Other Ig domains, such as the constant Ig domains in antibody heavy chains (e.g. CH1, CH2, and CH3) and light chains (C kappa, C lambda) may also be used as diversity scaffolds^(36, 37). Beyond antibodies and T cell receptors, the Ig domain has acted as a diversity scaffold expanding and evolving on an evolutionary timescale and Ig domains are found in many hundreds of different proteins involved in molecular recognition. Ig domains from such proteins may be used as diversity scaffold domains as described herein,

The scaffold of the diversity scaffold domain is the framework which maintains the secondary structural elements which combine to form the stable core structure of the domain. The scaffold comprises multiple contiguous or non-contiguous scaffold residues which form covalent and non-covalent interactions with the side chains or the peptide backbone of other scaffold residues in the domain. Interactions may include hydrogen bonds, disulphide bonds, ionic interactions and hydrophobic interactions. The scaffold residues may form secondary structural elements of the domain, such as a helices, 1 strands, 1 sheets, and other structural motifs. Because they contribute to the core structure of the diversity scaffold domain, scaffold residues are conserved and substitution of scaffold residues within the diversity scaffold domain is constrained.

Examples of scaffolds include the framework regions of an antibody variable domain or the cysteine knot framework of knottins (e.g. the six or more cysteine residues which form the characteristic knot structure) together with the knottin beta sheets and 3₁₀ helix motif.

The interaction sequence of a diversity scaffold domain is a contiguous or non-contiguous amino acid sequence which is presented by the stable core structure of the scaffold. The interaction sequence may interact with other molecules and mediate the binding activity of the domain, for example binding to a target molecule. In some embodiments, the interaction sequence of a domain may comprise one or more diverse residues, allowing selection of binding members with specific binding activity to be isolated from a library.

The residues of the interaction sequence may be entirely non-structural and may not support or contribute to the tertiary structure of the domain. For example, the interaction residues may be located at the loops or turns between secondary structural elements or motifs.

Because they do not contribute to the core structure of the diversity scaffold domain, the residues of the interaction sequence are less conserved and substitution of interaction residues within the diversity scaffold domain is less constrained. In some embodiments, the interaction sequence of a diversity scaffold domain may comprise residues in the non-loop faces of the protein^(43,44,52).

Examples of interaction sequences include the CDRs of an antibody variable domain and the loops joining secondary structural elements of stable, self-folding protein domains e.g. the joining loops of a knottin, such as the PRIL motif of loop 1 of EETI-II. Other examples of interaction sequences within scaffold domains are known in the art^(34-38, 41-48,51, 53).

In some embodiments, a domain may contain residues which contribute to both binding and secondary structure. These residues may constitute both scaffold and interaction sequences. This is illustrated with affibodies (the engineered Z domain of protein A domain)^(43, 52) and monobodies (engineered scaffolds based on a fibronectin domain)⁴⁴. Residues which contribute to both secondary structure and binding may be diversified in the binding members described herein. Binding members which retain secondary structure may, for example, be identified in libraries using routine selection techniques. In some embodiments, residues which contribute to both secondary structure and binding may not be diversified.

Diversity scaffold domains may be used as either donor or recipient diversity scaffold domains in the fusion proteins and binding members described herein.

The donor diversity scaffold domain comprises a donor scaffold and a donor interaction sequence.

Preferably, donor diversity scaffold domains have N and C termini that are proximal to each other within the native structure of the domain. The distance between the termini of the donor may, for example, be within 20% of the distance between the ends of the insertion site in the recipient diversity scaffold domain, more preferably the same distance. Examples of donor domains with proximal termini include knottins⁵⁴, adhirons⁴⁶ and Gp2 scaffolds⁵¹.

In other embodiments, donor diversity scaffold domains may have N and C termini that are not proximal in the native structure of the domain. Donor diversity scaffold domains with non-proximal N and C termini may be inserted into the recipient diversity scaffold domain as described herein using linkers that are sufficiently long to allow fusion of the termini of the donor domain with the loops of the recipient diversity scaffold domain, such that the linkers bridge the gap between the termini of donor and recipient domains. Alternatively, the donor diversity scaffold domain may be truncated to create a donor domain where the N and C termini are in proximity, or the recipient diversity scaffold domain may be truncated to create a recipient domain in which the termini of the insertion site are in proximity with the termini of the donor domain. The net result will be a loss of residues arising from the fusion of the donor and recipient structural domains. Linkers or truncations may reduce the distance between the termini of the donor domain to within 20% of the distance between the termini of the insertion site in the recipient scaffold or more preferably the same distance.

The donor diversity scaffold domain may consist of at least 15, at least 20, at least 25, at least 30 or at least 40 amino acids. The donor diversity scaffold domain may consist of 400 or fewer, 300 or fewer, 200 or fewer or 100 or fewer amino acids. For example, the donor diversity scaffold domain may consist of from 20 to 400 amino acids.

The donor diversity scaffold domain may comprise 2, 4, 6, 8, 10 or more cysteine residues which form disulphide bonds in the scaffold domain (i.e. the domain may contain 1, 2, 3, 4, 5 or more disulphide bonds).

In some embodiments, a donor diversity scaffolds may consist of at least 15 amino acids and contain 4 or more cysteine residues.

Examples of suitable donor diversity scaffold domains include an immunoglobulin, an immunoglobulin domain, a VH domain, a VL domain, an affibody, a cysteine-rich peptide, such as a venom toxin peptide or knottin, a “Designed ankyrin repeat protein” (DARPin), an adhiron, a fibronectin domain, an anticalin, a T7 phage gene 2 protein (Gp2) a monobody, a single domain antibody^(49, 50) or an affibody.

Preferred donor diversity scaffold domains include cysteine-rich peptides, such as ion channel-modulating peptides, venom toxin peptides and knottins.

Cysteine-rich peptides comprise a network of disulphide bonds as a core structural element. The structural conformations of cysteine-rich peptides are well-known in the art^(55, 6).

Ion channel-modulating peptides (both agonistic and antagonistic) comprising multiple disulphide bonds are well-known in the art. Examples include venom toxin peptides from venomous species, such as spiders, snakes, scorpions and venomous snails^(26, 55).

The structural conformations and disulphide linkage patterns of venom toxin peptides are also well known in the art. For example, an analysis of venoms of spiders and other animals reveals a multitude of conformations and patterns of disulphide linkage^(55, 65, 66, 67). For example, spider toxin huwentoxin-II has a disulphide linkage pattern of I-III, II-V, IV-VI⁶⁸ and “Janus-faced atracotoxins” (J-ACTXs) has a disulphide linkage pattern of I-IV, II-VII, III-IV and V-VIII (including an unusual “vicinal” disulphide bond between 2 neighbouring cysteines)⁵⁶, where the pairs of roman numeral refer to the order where each cysteine appears in the sequence and the position of the partner cysteine with which it forms a disulphide bond.

Cysteine-rich peptides may have a “disulphide-directed beta hairpin” (DDH) structure comprising an anti-parallel beta hairpin stabilised by 2 disulphide bonds⁵⁶. The DDH core structure is found in the widely studied “inhibitory cysteine knot” structure (hereafter referred to as cysteine-knot miniproteins or knottins).

Knottins are small cysteine rich proteins that have an interwoven disulfide-bonded framework, triple-stranded β-sheet fold, and one or more solvent exposed loops. Knottins typically comprise at least 3 disulfide bridges and are characterised by a disulphide knot which is achieved when a disulfide bridge between cysteines III and VI crosses the macrocycle formed by the two other disulfides (disulfides I-IV and II-V) and the interconnecting backbone. These bridges stabilise the common tertiary fold formed of antiparallel β-sheets and in some cases a short 3₁₀ helix. (The pairs of Roman numeral refers to the order where each cysteine appears in the sequence and the position of the partner cysteine with which it forms a disulphide bond). Knottins are 20-60 residues long, usually 26-48 residues, and are found in diverse organisms ranging from arthropods, molluscs, and arachnids to plants⁵⁷. Knottins arising from conus snails (conotoxins) in particular have been widely studied^(54,58-60). Many thousands of other knottins have been identified and their sequences and structures are publically available^(57,61,62) for example from on-line databases (such as the Knottin on-line database⁵⁷, Centre de Biochimie Structural, CNRS, France).

In some embodiments, the overall correct secondary structure of the knottin may be conferred by the correct distribution and spacing of cysteines. In other embodiments one or more additional scaffold residues may be required to confer the correct secondary structure. For example, residues 11-15 and 22-25 of EETI-II direct the folding propensity towards the 11-15 3₁₀-helix region and a 22-25 β-turn region respectively in the absence of any disulphides⁶³.

Knottins display a high degree of sequence flexibility and can accommodate large amounts of non-native sequence. For example, EETI-II can accommodate over 50% non-native sequence (by randomising 2 of the loops)²⁴.

A suitable cysteine-rich peptide for use as a diversity scaffold as described herein may for example comprise the amino acid sequence of Huwentoxin-IV, ProTx-II, Ssm6a, Kaliotoxin, mokatoxin-1, Conotoxin-ω, MCoTI-II, Shk, PcTX1 or mambalgin (as shown in Table 8A; SEQ ID NOS: 7-25) or other sequence set out in the Knottin database or may be a fragment or variant of this sequence which retains the correct fold structure.

A knottin which is a variant of a reference knottin sequence, such as sequence shown in Table 8A (SEQ ID NOS: 7-25) or set out in the Knottin database, may comprise an amino acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the reference sequence. Particular amino acid sequence variants may differ from a knottin sequence of Table 8A (SEQ ID NOS: 7-25) by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 amino acids.

Sequence similarity and identity are commonly defined with reference to the algorithm GAP (Wisconsin Package, Accelerys, San Diego USA). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty=12 and gap extension penalty=4. Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990) J. Mol. Biol. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988) PNAS USA 85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981) J. Mol Biol. 147: 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res. (1997) 25 3389-3402) may be used.

Sequence comparison may be made over the full-length of the relevant sequence described herein.

One or more residues within a loop of a knottin, for example, one or more residues within loop 1, 2, 3, 4 or 5 of a knottin may be diversified or randomised. In some embodiments, one or more within the target binding motif, such as the trypsin binding motif PRIL in loop 1 of EETI-II, or the corresponding residues in a different knottin, may be diversified. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more residues may be diversified.

The formation of native disulphide linkage patterns in the donor and recipient diversity together and the adoption of correct secondary structures within the scaffold domains of a fusion protein is exemplified below.

In other preferred embodiments, the donor diversity scaffold is an adhiron. Adhirons are peptides of about 80-100 amino acids based on plant-derived phytocystatins⁴⁶. Suitable adhiron sequences are well-known in the art and described elsewhere herein. A suitable adhiron for use as a diversity scaffold as described herein may for example comprise an amino acid sequence shown in Table 12 (SEQ ID NOS: 26, 27) or may be a fragment or variant of this sequence.

An adhiron which is a variant of a reference adhiron sequence, such as sequence shown in Table 12 (SEQ ID NOS: 26 & 27) may comprise an amino acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the reference sequence. Particular amino acid sequence variants may differ from a sequence of Table 12 (SEQ ID NOS: 26 & 27) by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 amino acids.

The methods described herein do not require knowledge of the structure of the donor diversity scaffold domain. Selection of binding members may be based on binding to the target molecule (if known) or the correct folding of the recipient domain. The donor diversity scaffold domain may be provided within a library from which clones with proper folding of the recipient scaffold can be selected or may be inserted into an existing recipient diversity scaffold which accommodates an incoming donor diversity scaffold.

The failure of one scaffold domain to fold will affect the overall expression and stability of the resultant fusion protein and so it will also be possible to introduce diversification in the absence of structural knowledge and screen for retained expression of a folded scaffold domain. However, where the structure of the donor diversity scaffold domain is known, sites for diversification in the construction of libraries may be guided by this structure.

The recipient diversity scaffold domain and the donor diversity scaffold domain are preferably heterologous i.e. they are associated by artificially by recombinant means and are not associated in nature. In some preferred embodiments, the donor and recipient diversity scaffold domains are from different scaffold classes e.g. they are not both immunoglobulins, both cysteine-rich proteins, both knottins or both adhirons.

The recipient diversity scaffold domain comprises a recipient scaffold and a recipient interaction sequence.

In some embodiments, the recipient diversity scaffold domain may lack cysteine residues which form disulphide bonds. For example, the scaffold domain may comprise 0 or 1 cysteine residue. In other embodiments, the recipient diversity scaffold domain may comprise 2, 4, 6, 8, 10 or more cysteine residues which form disulphide bonds in the scaffold domain (i.e. the domain may comprise 1, 2, 3, 4, 5 or more disulphide bonds).

Examples of suitable recipient diversity scaffold domains include an immunoglobulin domain, a VH domain, a VL domain, a knottin, a Protein A, cysteine-rich peptide, venom toxin, a Designed ankyrin repeat protein (DARPin), an adhiron, a fibronectin domain, an anticalin and a T7 phage gene 2 protein (Gp2).

Selection of the insertion site for the donor diversity scaffold domain and linker sequences may be guided by the structure of the recipient domain, where the structure is known^(64, 53, 23, 75, 39, 43). For example, a suitable insertion site may be located in a region which joins secondary structural elements of the recipient diversity scaffold domain, such as beta strands or alpha helices. Suitable regions for the insertion site within the 10^(th) type III cell adhesion domain of fibronectin are shown in FIG. 20A (SEQ ID NO: 28) and regions for the insertion site within the T7 Gp2 protein are shown in FIG. 20B (SEQ ID NO: 29). The identification of suitable insertion sites is described in more detail in example 10 below. Structural knowledge may also be used to direct diversification in the construction of libraries.

Preferably, the recipient diversity scaffold domain is all or part of an immunoglobulin, most preferably, all or part of an antibody variable domain. For example, the recipient diversity scaffold domain may be an antibody light chain variable (VL) domain or an antibody heavy chain variable (VH) domain.

The incoming donor diversity scaffold domain separates the recipient diversity scaffold domain into N terminal and C terminal parts at the point of insertion into the recipient. The donor diversity scaffold domain is fused internally within the recipient diversity scaffold domain such that the N terminus and C terminus of the donor diversity scaffold domain are connected either directly or via a linker to the N and C terminal parts respectively of the recipient diversity scaffold domain. In some embodiments, one or more residues at the N and/or C terminals of the donor diversity scaffold domain or the recipient diversity scaffold domain may be removed and/or randomised. The recipient diversity scaffold domain retains its original N and C termini, which are not affected by the insertion of the donor diversity scaffold domain into the internal insertion site.

In some embodiments, the orientation of the incoming donor diversity scaffold domain relative to the recipient diversity scaffold domain may be altered by linking the recipient diversity scaffold domain to different positions within the donor diversity scaffold domain. For example, a rotated donor diversity scaffold domain may be designed by cyclising the donor diversity scaffold domain through linkage of the native N and C terminals and linearizing at a different position in the amino acid sequence to generate artificial N and C terminals. These artificial terminals may be linked to the recipient diversity scaffold domain within the fusion protein. In other words, the native N and C termini of a donor diversity scaffold domain, such as a cysteine-rich peptide, may be joined together directly or via a peptide sequence and artificial N and C termini generated at a different position in the sequence of the donor diversity scaffold domain. In a fusion protein generated using these artificial N and C termini, the donor diversity scaffold domain is rotated relative to the recipient diversity scaffold domain compared to a fusion protein comprising the donor diversity scaffold domain with native N and C terminals. For example, the order of the loops within the donor diversity scaffold domain may be altered. Examples of EETI-II donor diversity scaffold domains with altered orientation are shown in Table 28 (SEQ ID NOS: 302-307).

Preferably, the donor diversity scaffold domain completely replaces some or all of a loop sequence of the recipient diversity scaffold domain, such that the donor diversity scaffold domain is directly linked to scaffold residues of the recipient diversity scaffold domain. For example, loop sequences joining secondary structural elements in the recipient domain (e.g. a CDR joining 2 β strands of a β sheet framework elements in an antibody variable domain) may be removed in their entirety. In some embodiments, one or more scaffold residues may be removed or replaced from the donor diversity scaffold domain or the recipient diversity scaffold domain to reduce the distance between them while enabling folding of the component parts of the fusion protein.

Preferably, the donor diversity scaffold domain is positioned in the fusion protein close to the interaction sequence of the recipient diversity scaffold domain. For example, donor diversity scaffold may be located less than 20 Angstroms from the recipient donor scaffold, for example 8-13 Angstroms in the case of the knottin insertion exemplified herein; calculated from the end of the preceding framework to the first cysteine of the donor/knot motif. This proximity allows both interaction sequences to interact with the same or closely apposed sites on the target molecule or complex, such that both donor and recipient domains may contribute simultaneously to binding. For example, the fusion protein may lack rigid stalks and flexible linkers to connect the donor diversity scaffold domain to the recipient diversity scaffold domain.

In some preferred embodiments, the recipient diversity scaffold is an antibody variable domain, for example an antibody VH or VL domain.

The scaffold of a VH, VL kappa or VL lambda recipient domain may comprise residues 1-26, 39-55 and 66-104 (IMGT numbering) of the domain. The scaffold may also comprise residues of framework 4 which are encoded by the last 11 residues encoded by the J segments of the heavy chain and by the last 10 residues encoded by the J segments of the kappa and lambda light chains. The interaction sequence of a VH, VL kappa or VL lambda domain may comprise residues 27 to 38 (CDR1), 56 to 65 (CDR2) and 105 to 117 (CDR3) (IMGT numbering).

Examples of preferred recipient diversity scaffold domains are shown in Table 1 with inserted donor diversity scaffold domains.

Other preferred recipient scaffolds include antibody constant domains, for example a heavy or light chain CH1, CH2 or CH3 domain.

The donor diversity scaffold domain may replace part or more preferably all of a CDR (i.e. CDR1, CDR2 or CDR3) in the recipient antibody variable domain.

Preferably, the donor diversity scaffold domain replaces all or part of the CDR1 or CDR2 of the antibody variable domain. This allows the more diverse and central CDR3 to contribute to the binding activity, as well as the partner antibody variable domain. Insertion of the donor diversity scaffold domain into CDR1 and CDR2 is exemplified below.

The interaction sequence of the donor diversity scaffold domain and one or more CDRs of the recipient antibody variable domain or partner domain may interact with the same epitope on the target molecule. For example, the interaction sequence of the donor diversity scaffold domain and the CDR3 of the antibody variable domain and/or partner domain may interact with the same epitope on the target molecule.

In some preferred embodiments, the recipient diversity scaffold domain is an antibody variable domain, for example a VH or VL domain, and the donor diversity scaffold domain is a knottin or adhiron. Examples of binding members comprising a knottin donor diversity scaffold domain and a VL domain recipient diversity scaffold are shown in Table 1. A binding member comprising an antibody variable domain recipient and a cysteine rich donor domain is termed a “Knotbody” herein.

In one set of preferred embodiments, the recipient diversity scaffold domain is an antibody VL domain, and the donor diversity scaffold domain is a knottin which replaces the CDR2 of the VL domain. Suitable knottins may bind to an ion channel, such as Kv1.3, and may include ShK and Kaliotoxin. The recipient antibody VL domain may show no target binding or may bind to the same target molecule as the knottin, an associated protein in complex with the knottin target molecule or a different target molecule to the knottin.

A suitable binding member may for example comprise a fusion protein having the amino acid sequence of any one of SEQ ID NOS: 31 to 35 (as shown in Table 8B) or may be a fragment or variant of such a sequence. The binding member may further comprise a partner domain which associates with the fusion protein. Suitable partner domains include any VH domain, for example the D12 A12 VH domain shown in Table 8B (SEQ ID NO: 30).

Suitable VH domains may be non-binders, may bind to a different target molecule to the fusion protein or may bind to the same target molecule as the fusion protein, for example to increase binding affinity of the fusion protein.

A fusion protein which is a variant of a reference sequence, such as sequence shown in Table 1 (SEQ ID NOS: 84-139) Table 8B (SEQ ID NOS: 31 to 35), Table 29 (SEQ ID NOS: 308-317) or FIG. 29 (SEQ ID NOS 349 and 351) may comprise an amino acid sequence having at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 98% sequence identity to the reference sequence. Particular amino acid sequence variants may differ from a fusion protein sequence of Table 1 (SEQ ID NOS: 84-139) Table 8B (SEQ ID NOS: 31 to 35), Table 29 (SEQ ID NOS: 308-317) or FIG. 29 (SEQ ID NOS 349 and 351) by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more than 10 amino acids.

In other preferred embodiments, the recipient diversity scaffold domain may be an immunoglobulin constant domain, for example an immunoglobulin constant domain from an antibody heavy or light chain, and the donor diversity scaffold domain may be a knottin or adhiron.

Preferably, the distance between the framework residues of the recipient diversity scaffold domain and the donor diversity scaffold domain is minimised to reduce proteolytic lability and/or relative flexibility between the domains. This may be useful in promoting binding affinity. For example, the N and C terminals of the donor diversity scaffold domain may each be linked directly to the recipient diversity scaffold domain without a linker or through linkers of 1, 2, 3 or 4 amino acids. The same or different linker sequences may be used to link the N and C terminals of the donor diversity scaffold domain to the recipient diversity scaffold domain.

Examples of suitable linker sequences are shown in Tables 2, 26 and 27.

In some embodiments, it may be preferred that the donor and recipient diversity scaffold domains are joined by a linker other than GGSG (SEQ ID NO: 2), a linker other than GGGS (SEQ ID NO: 32) or a linker that does not comprise GG, such as SGG or GG.

Linker length refers to the number of amino acid residues that are gained or lost from the scaffold elements of the donor and recipient domains when fused together. For example, the removal of scaffold residues and the use of an equal number of randomized residues to join the donor and recipient diversity scaffold domains is considered herein to be randomization of the removed framework residues rather than the introduction of additional linker residues.

In some embodiments, 1, 2, 3 or 4 amino acids at the N and/or C terminal of the donor diversity scaffold domain and/or 1, 2, 3 or 4 amino acids on each side of the insertion site in the recipient scaffold may be replaced through mutagenesis and/or randomisation.

The fusion protein may be associated with the partner domain through a covalent or non-covalent bond.

The partner domain may be associated with the recipient diversity scaffold domain and/or the donor diversity scaffold domain of the fusion protein. Preferably, the partner domain is associated with the recipient diversity scaffold domain.

In some preferred embodiments, the recipient diversity scaffold domain is an antibody light chain variable (VL) domain and the partner domain is an antibody heavy chain variable (VH) domain. In other preferred embodiments, the recipient diversity scaffold domain is an antibody heavy chain variable (VH) domain and the partner domain is an antibody light variable (VL) domain.

In some embodiments, antibody heavy and light chain variable domains of a binding member as described herein may be connected by a flexible linker, for example in a scFv format.

A binding member described herein comprises the fusion protein. The binding member may further comprise a partner domain that is associated with the fusion protein. For example, the binding member may be a heterodimer.

In some embodiments, the binding member may comprise two or more fusion proteins or partner domains. For example, the binding member may be multimeric. In some embodiments, the recipient or donor diversity scaffold domain of the binding member may form a homomultimer, such as a homodimer. The recipient or donor diversity scaffold domain of the binding member may partner with a different form of the same domain, such as a wild-type or mutagenized form of the domain.

In addition to the fusion protein and optional partner domain, the binding member may further comprise a therapeutic moiety, half-life extension moiety or detectable label. The moiety or label may be covalently or non-covalently linked to the fusion protein or the partner domain.

In some embodiments, a binding member may be displayed on a particle or molecular complex, such as a cell, ribosome or phage, for example for screening and selection. The binding member may further comprise a display moiety, such as phage coat protein, to facilitate display on a particle or molecular complex. The phage coat protein may be fused or covalently linked to the fusion protein or the partner domain.

In some embodiments, a binding member as described herein may display advantageous properties, such as increased affinity, stability, solubility, expression, specificity or in vivo half-life relative to the isolated donor and/or recipient diversity scaffold domain.

Other aspects of the invention relate to the generation of libraries of binding members as described herein.

A method of producing a library of binding members may comprise;

-   -   providing a population of nucleic acids encoding a diverse         population or repertoire of fusion proteins comprising a donor         diversity scaffold domain inserted into a recipient diversity         scaffold domain, optionally wherein the N and C terminals of the         donor diversity scaffold domain are linked to the recipient         diversity scaffold domain with linkers of up to 4 amino acids,     -   wherein the donor diversity scaffold domain comprises a donor         scaffold and a donor interaction sequence and the recipient         diversity scaffold domain comprises a recipient scaffold and a         recipient interaction sequence, and     -   one, two or all three of the donor interaction sequence, the         recipient interaction sequence and the linkers are diverse in         said population, and     -   expressing said population of nucleic acids to produce the         diverse population or repertoire, and     -   optionally associating the fusion proteins with a partner domain         or population of partner domains, thereby producing a library of         binding members.

The population of nucleic acids may be provided by a method comprising inserting a first population of nucleic acids encoding a donor diversity scaffold domain into a second population of nucleic acids encoding a recipient diversity scaffold domain, optionally wherein the first and second nucleic acids are linked with a third population of nucleic acids encoding linkers of up to 4 amino acids, wherein the one, two or all three of the first, second and third populations of nucleic acids are diverse.

The nucleic acids may be contained in vectors, for example expression vectors. Suitable vectors include phage-based⁷⁶ or phagemid-based⁷⁷ phage display vectors.

The nucleic acids may be recombinantly expressed in a cell or in solution using a cell-free in vitro translation system such as a ribosome, to generate the library. In some preferred embodiments, the library is expressed in a system in which the function of the binding member enables isolation of its encoding nucleic acid. For example, the binding member may be displayed on a particle or molecular complex to enable selection and/or screening. In some embodiments, the library of binding members may be displayed on beads, cell-free ribosomes, bacteriophage, prokaryotic cells or eukaryotic cells. Alternatively, the encoded binding member may be presented within an emulsion where activity of the binding member causes an identifiable change. Alternatively, the encoded binding member may be expressed within or in proximity of a cell where activity of the binding member causes a phenotypic change or changes in the expression of a reporter gene^(16, 78, 79).

Eukaryotic cells benefit from chaperone systems to assist the folding of secreted protein domains⁸⁰ and these are absent within bacterial expression systems. The data set out below shows that mammalian chaperone systems are not required for the correct folding of the binding members described herein. In addition, binding members identified using prokaryotic phage display systems are by definition amenable to further engineering within the phage display system to create improved binders. Prokaryotic systems, such as phage display, also facilitate construction of larger libraries and facilitate selection, screening and identification of binding members from such libraries. In contrast binding members identified in eukaryotic systems may not be amenable for further engineering within prokaryotic systems.

Preferably, the nucleic acids are expressed in a prokaryotic cell, such as E coli. For example, the nucleic acids may be expressed in a prokaryotic cell to generate a library of binding members that is displayed on the surface of bacteriophage. Suitable prokaryotic phage display systems are well known in the art, and are described for example in Kontermann, R & Dubel, S, Antibody Engineering, Springer-Verlag New York, LLC; 2001, ISBN: 3540413545, WO92/01047, U.S. Pat. Nos. 5,969,108, 5,565,332, 5,733,743, 5,858,657, 5,871,907, 5,872,215, 5,885,793, 5,962,255, 6,140,471, 6,172,197, 6,225,447, 6,291,650, 6,492,160 and 6,521,404. Phage display systems allow the production of large libraries, for example libraries with 10⁸ or more, 10⁹ or more, or 10¹⁰ or more members.

In other embodiments, the cell may be a eukaryotic cell, such as a yeast, insect, plant or mammalian cell.

A library of binding members, for example a library generated by a method described above, may comprise;

-   -   a diverse population of fusion proteins comprising a donor         diversity scaffold domain inserted into a recipient diversity         scaffold domain, optionally wherein the N and C terminals of the         donor diversity scaffold domain are each linked to the recipient         diversity scaffold domain with linkers of up to 4 amino acids,     -   wherein the donor diversity scaffold domain comprises a donor         scaffold and a donor interaction sequence and the recipient         diversity scaffold domain comprises a recipient scaffold and a         recipient interaction sequence, and     -   one, two or all three of the donor interaction sequence, the         recipient interaction sequence and the linkers are diverse in         said population, and     -   optionally wherein each fusion protein in the population is         associated with a partner domain.

The sequences of one, two or all three of the donor interaction sequence, the recipient interaction sequence and the linkers may be diverse. For example, the binding members in the library may have diverse donor interaction sequences and the same recipient interaction sequence; diverse recipient interaction sequences and the same donor interaction sequence; diverse linker sequences and the same recipient and donor interaction sequences or; diverse recipient and donor interaction sequences.

A diverse sequence as described herein is a sequence which varies between the members of a population i.e. the sequence is different in different members of the population. A diverse sequence may be random i.e. the identity of the amino acid or nucleotide at each position in the diverse sequence may be randomly selected from the complete set of naturally occurring amino acids or nucleotides or a sub-set thereof.

Diversity may be targeted to donor interaction sequence, recipient interaction sequence, linkers or sequences within the partner domain using approaches known to those skilled in the art, such as oligonucleotide-directed mutagenesis^(22, 23) , Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press, and references therein). For example, where the recipient diversity scaffold domain is an antibody variable domain and the donor diversity scaffold domain has replaced one CDR region, amino acid changes, such as diverse sequences, may be introduced into the other CDRs of the recipient domain or partner chain. Where the donor domain is a knottin, diverse sequences may be introduced into regions linking core scaffold elements such as the cysteine knot. The knottin structure has been shown to be able to accommodate over 50% non-native structure²⁴. The introduction of diversity into the knottin scaffolds, such as EETI-II²⁴, kaliotoxin²⁵, ProTx-1²⁶, and TRPA1²⁶ to generate libraries is known in the art.

Diverse sequences may be contiguous or may be distributed within the domain. Suitable methods for introducing diverse sequences into domains are well-described in the art. For example, diversification may be generated using oligonucleotide mixes created using partial or complete randomisation of nucleotides or created using codons mixtures, for example using trinucleotides. Alternatively, a population of diverse oligonucleotides may be synthesised using high throughput gene synthesis methods and combined to create a precisely defined and controlled population of variant domains. Alternatively “doping” techniques in which the original nucleotide predominates with alternative nucleotide(s) present at lower frequency may be used. As an alternative, Example 2 below describes methods for introducing diversity into a domain using diversity from a natural source (an antibody repertoire in this example).

Either donor or recipient domains could be part of a multimeric protein with potential additional diversity being introduced by the partner chain. The entire partner chain can be replaced e.g. by chain shuffling²⁷. Alternatively, diversity could be introduced into regions of an existing partner chain of the recipient or donor e.g. the VH partner of a VL recipient of the donor. In general the heterodimeric nature of antibodies and the ability to select variants of the partner chain independently of the recipient chain supporting the donor adds to the power of this approach.

Preferably, the library is a display library. The binding members in the library may be displayed on the surface of particles, or molecular complexes such as beads, ribosomes, cells or viruses, including replicable genetic packages, such as yeast, bacteria or bacteriophage (e.g. Fd, M13 or T7) particles, viruses, cells, including mammalian cells, or covalent, ribosomal or other in vitro display systems. Each particle or molecular complex may comprise nucleic acid that encodes the binding member that is displayed by the particle and optionally also a displayed partner domain if present.

In some preferred embodiments, the binding members in the library are displayed on the surface of a bacteriophage. Suitable methods for the generation and screening of phage display libraries are well known in the art. Phage display is described for example in WO92/01047 and US patents U.S. Pat. Nos. 5,969,108, 5,565,332, 5,733,743, 5,858,657, 5,871,907, 5,872,215, 5,885,793, 5,962,255, 6,140,471, 6,172,197, 6,225,447, 6,291,650, 6,492,160 and 6,521,404.

Libraries as described herein may be screened for binding members which display binding activity, for example binding to a target molecule.

Binding may be measured directly or may be measured indirectly through agonistic or antagonistic effects resulting from binding.

Binding to the target molecule may be mediated by one or more of the donor diversity scaffold domain, the recipient diversity scaffold domain and the partner domain of the binding member.

The interaction sequences of the donor and recipient diversity scaffold domains of the fusion protein may bind to the target molecule, and more preferably the same epitope of the target molecule. The fusion protein and the partner domain of a binding member may bind to the same target molecule or different target molecules. For example, the fusion protein may bind to a first target molecule and the partner domain may bind to a second target molecule.

A binding member may bind the target molecule with the same affinity of a parent donor or recipient diversity scaffold domain, or with a lower or higher affinity. In some embodiments, a binding member may neutralise a biological activity of the target molecule. In other embodiments, a binding member may activate a biological activity of the target molecule.

Suitable target molecules include biological macromolecules, such as proteins. The target molecule may be a receptor, enzyme, antigen or oligosaccharide. In some preferred embodiments, the target may be an integral membrane protein. In some embodiments, the target may be G protein coupled receptor (GPCR). Target molecules which are difficult to target with antibodies, such as ion transporters, may be particularly suitable.

In some preferred embodiments, the target molecule may be an ion transporter, such as an ion pump or ion channel.

Suitable ion channels are well known in the art and include Kir1.1, Kir2.1, Kir6.2, SUR2, Kv1.1, KCNQ1, KCNQ, KCNQ4, TRPP2, TRPA1, TRPC6, CNGA1, BK, Nav1.1, Nav1.5, Nav1.6, Nav2.1, Cav1.2, Cav2.1, glycine receptors, GABA-A, CHRNA4⁸² Other suitable ion channels include voltage-gated potassium channels such as Kv1.3, L-Type voltage-gated calcium channels, Cav2.2, hERG, ASICs and Eag1.

A binding member described herein may bind to two or more different target epitopes (i.e. the binding member may be multi-specific). The target epitopes bound by the binding member may be on the same or different target molecules. For example, one or more of the donor diversity scaffold domain, recipient diversity scaffold domain and/or partner domain of a multi-specific binding member may bind to a different target epitope to the other domains.

In some embodiments, the binding member may be bi-specific i.e. it may bind to two different epitopes.

A bi-specific binding member may bind to two different target epitopes concurrently. This may be useful in bringing a first and a second epitope into close proximity for example to cross-link molecules or cells. When the target epitopes are located on different target molecules, the target molecules may be brought into close proximity by concurrent binding to the binding member. When the target molecules are located on different cells, concurrent binding of the target molecules to the binding member may bring the cells into close proximity, for example to promote or enhance the interaction of the cells. For example, a binding member which binds to a tumour specific antigen and a T cell antigen, such as CD3, may be useful in bringing T cells into proximity to tumour cells.

Concurrent binding of different target epitopes by a binding member described herein may also be useful in stabilizing a particular conformation of a target molecule or complex of target molecules.

A binding member may bind to the two different target epitopes sequentially i.e. the binding member may bind to one target epitope and then the other. This may be useful, for example in crossing the blood: brain barrier (BBB¹²⁰) or blood: nerve barrier (BNB¹²¹). For example, a binding member may bind to an epitope on a BBB or BNB receptor. Suitable receptors are well-known in the art and include transferrin (TFR), insulin (IR), leptin (LEP-R), glucose (GLUT1), CD98 (LAT1) and lipoprotein (LRP-1) receptors. Once bound to the BBB or BNB receptor, the binding member may be transcytosed across the BBB or BNB, after which it may then bind independently to an epitope on a second target molecule in the brain or peripheral nervous system, for example an ion channel or protease.

In some preferred embodiment, a binding member may bind to TFR. For example, the binding member may comprise a partner domain that binds to TFR and a fusion protein that binds to a second target molecule in the brain or peripheral nervous system. The recipient scaffold in the fusion protein may be a VL domain and the partner domain may be a VH domain. Suitable VH partner domains for targeting TFR may comprise a sequence shown in Table 20 (SEQ ID NOs: 237-245) or a variant thereof.

Suitable in vitro systems for screening libraries for bi-specific binding members able to cross the BBB or BNB are well known in the art (Nakagawa, S. et al. Neurochem. Int. 54, 253-263 (2009); Yosef, N. et al J. Neuropathol. Exp. Neurol. 69, 82-97 (2010)) and described in more detail below.

In some preferred embodiments, a first domain on the binding member (e.g. one of the donor diversity scaffold domain, recipient diversity scaffold domain and partner domain) may be directed to a first cell surface target molecule. Binding to this first target molecule may sequester the binding member on the cell surface, thereby facilitating interaction of a second binding domain (e.g. another of the donor diversity scaffold domain, recipient diversity scaffold domain and partner domain) to a second target molecule by increasing the local concentration of the binding member on the cell surface. For example, A bi-specific binding member may consist of a VH partner domain which binds with high affinity to an abundant first target molecule on T cells coupled with a VL-knottin fusion which binds and blocks a second target molecule, such as an ion channel e.g. Kv1.3. A cell-specific binding domain (VH in this example) may be generated, for example, by selecting on recombinant protein representing a candidate target molecule (as described in example 6) or by selecting a library on the cell surface of a target cell to look for a partner domain (VH in this example) which enhances the binding of the second domain. Binding of the first domain to the first target molecule sequesters the binding member on the cell surface and may overcome low affinity, low density or relative inaccessibility of the second binding domain for the second target molecule. This may increase the potency of the second binding domain on cells which express the target recognised by the first binding domain. This approach may also increase the specificity of the binding member, since optimal binding/blocking require both target molecules to be on the same cell. The presence of a first binding molecule has been shown to effect a binding enhancement of a second binding molecule over several orders of magnitude based on affinity and antigen density (Rhoden et al (2016) J Biol. Chem. 291 p 11337-11347).

Target molecules for use in selection and screening may be produced using any convenient technique. For example, integral membrane proteins may be produced using bacterial, baculoviral or mammalian expression systems^(86,87), proteoliposomes⁸⁸, “nanodiscs”⁸⁹ or cell lysates⁹⁰.

In some embodiments described herein, an initial “founder” library of binding members may be used to identify one or more founder binding members which allow simultaneous folding of both donor and recipient diversity scaffold domains. These founder binding members may be selected for retained donor and recipient domains which display the desired binding activity, such as binding to a target molecule. In subsequent steps, the founder binding members may be subjected to further modification and screening to optimise binding activity and other properties. Alternatively, an initial “founder” library of binding members may include any library that combines a recipient diversity scaffold domain, a donor diversity scaffold domain and, optionally a linker, as described herein, in which diversity is introduced into any of said recipient diversity scaffold domain, a donor diversity scaffold domain or linker sequence.

A method of screening as described herein may comprise;

-   -   (i) providing a founder library of binding members as described         herein;     -   wherein one, two or all three the donor interaction sequence,         the recipient interaction sequence and the linkers are diverse         in said founder library,     -   (ii) screening the library for binding members which display a         binding activity, and     -   (iii) identifying one or more binding members in the founder         library which display the binding activity.

The one or more identified binding members may be recovered from the founder library.

In some preferred embodiments, the linker sequences may be diverse in the library. This may be useful in identifying functional fusions of donor and recipient diversity scaffold domains. Examples of diverse linker sequences are presented herein. In some embodiments, diverse linkers of 1, 2, 3, or 4 amino acids may be introduced at the junctions between the donor diversity scaffold domain and the recipient diversity scaffold domain to allow selection of optimal junctional sequences from the resultant libraries.

Optionally, sequences within the recipient diversity scaffold domain may also be diversified to facilitate the identification of a recipient diversity scaffold domain that supports functional fusion to a donor diversity scaffold domain. In some embodiments, one or more amino acid residues of the donor diversity scaffold domain and/or recipient diversity scaffold domain at one or both junctions between the two domains may be diversified (i.e. one or more residues of a domain directly adjacent one or both junctions with the other domain may be diversified). Examples in which framework residues of donor and recipient scaffolds are diversified are provided below. For example, 1, 2, 3, or 4 residues at the N and C terminal junctions of the donor diversity scaffold domain and/or 1, 2, 3, or 4 residues within the recipient diversity scaffold domain at the junctions with the donor domain may be diversified.

The binding activity may be binding to a target molecule. A method of screening a library may comprise;

-   -   providing a founder library of binding members as described         herein,     -   contacting the founder library with a target molecule, and     -   selecting one or more founder library members which bind to the         target molecule.

The one or more identified or selected binding members may be recovered and subjected to further selection and/or screening.

Multiple rounds of panning may be performed in order to identify binding members which display the binding activity. For example, a population of binding members enriched for the binding activity may be recovered or isolated from the founder library and subjected to one or more further rounds of screening for the binding activity to produce one or more further enriched populations. Founder binding members which display binding activity may be identified from the one or more further enriched populations and recovered, isolated and/or further investigated.

Founder binding members which display the binding activity may be further engineering to improve activity or properties or introduce new activity or properties, for example binding properties such as affinity and/or specificity, increased neutralization of the target molecule and/or modulation of a specific activity of the target molecule. Binding members may also be engineered to improve stability, solubility or expression level.

For example, the identified linker sequence of an identified founder binding member may be retained in a modified library with diverse sequences introduced into one or more of the recipient, donor or partner domains depending on the application and desired outcome. In some cases further diversification of the linker may be useful in optimising function of the binding member.

A method of screening as described herein may further comprise;

-   -   (iv) introducing diverse amino acid residues at one or more         positions in the amino acid sequence of one or more binding         members as described herein, for example one or more binding         members identified in from a founder library, to produce a         modified library of binding members,     -   (v) screening the modified library for modified binding members         which display a binding activity and     -   (vi) identifying one or more modified binding members in the         modified library which display the binding activity.

The binding activity may be binding to a target molecule. A method of screening a library may comprise;

-   -   providing a modified library of binding members as described         above,     -   contacting the modified library with a target molecule and     -   selecting one or more modified library members which bind to the         target molecule.

The one or more identified or selected binding members may be recovered and subjected to further rounds of screening.

The diverse amino acids may be introduced by any suitable technique as described elsewhere herein, including random mutagenesis or site directed mutagenesis.

One or more modified binding members may be identified which display increased or improved binding activity, for example increased binding affinity and/or specificity, relative to the one or more founder binding members identified in step (iii).

Multiple rounds of panning may be performed in order to identify modified binding members which display the improved binding activity. For example, a population of binding members enriched for the improved binding activity may be recovered or isolated from the library and subjected to one or more further rounds of screening for the binding activity to produce one or more further enriched populations. Modified binding members which display improved binding activity may be identified from the one or more further enriched populations and isolated and/or further investigated.

Founder or modified binding members may be further subjected to further mutagenesis, for example to generate further modified libraries and select binding members with improved or new activity or properties. Amino acid residues may be mutated at one or more positions in the amino acid sequence of one or more identified binding members from the library and/or modified library. For example, amino acid residues within the donor scaffold, donor interaction sequence, recipient scaffold, recipient interaction sequence, linker or partner domain of the one or more identified binding members may be mutated.

The mutation may introduce diverse amino acid residues at the one or more positions to produce a library of further modified binding members. Examples of the deletion or replacement of interaction and scaffold residues of recipient and donor and their replacement with randomised residues are shown below.

To generate binding members in which a partner domain, such as a partner VH or VL domain, contributes additional interaction contacts with a target molecule, a library may be generated in which the original partner domain is replaced by a whole repertoire of alternative partner domain to create a “chain-shuffled” library. Binding members with improved activity due to the beneficial contribution of the newly selected partner domain may be identified, recovered and isolated.

A method of screening as described herein may further comprise;

-   -   (vii) associating the fusion protein from the one or more         identified binding members or modified binding members as         described above, with a diverse population of partner domains to         produce a shuffled library of binding members,     -   (viii) screening the shuffled library for shuffled binding         members which display a binding activity,     -   (ix) identifying one or more shuffled binding members which         display the binding activity.

One or more shuffled binding members may be identified which display increased binding activity relative to the one or more founder binding members identified in step (iii) and/or modified binding members identified in step (vi).

Multiple rounds of panning or functional screening may be performed in order to identify shuffled binding members which display the increased or improved binding activity. For example, a population of shuffled binding members enriched for the improved binding activity may be isolated from the shuffled library and subjected to one or more further rounds of screening for the binding activity to produce one or more further enriched shuffled populations. Shuffled binding members which display improved binding activity may be identified from the one or more further enriched populations and isolated and/or further investigated.

The founder library, modified library or shuffled library may be screened by determining the binding of the binding members in the library to a target molecule Binding may be determined by any suitable technique. For example, the founder library, modified library or shuffled library may be contacted with the target molecule under binding conditions for a time period sufficient for the target molecule to interact with the library and form a binding reaction complex with a least one member thereof.

Binding conditions are those conditions compatible with the known natural binding function of the target molecule. Those compatible conditions are buffer, pH and temperature conditions that maintain the biological activity of the target molecule, thereby maintaining the ability of the molecule to participate in its preselected binding interaction. Typically, those conditions include an aqueous, physiologic solution of pH and ionic strength normally associated with the target molecule of interest.

The founder library, modified library or shuffled library may be contacted with the target molecule in the form of a heterogeneous or homogeneous admixture. Thus, the members of the library can be in the solid phase with the target molecule present in the liquid phase. Alternatively, the target molecule can be in the solid phase with the members of the library present in the liquid phase. Still further, both the library members and the target molecule can be in the liquid phase.

Suitable methods for determining binding of a binding member to a target molecule are well known in the art and include ELISA, bead-based binding assays (e.g. using streptavidin-coated beads in conjunction with biotinylated target molecules, surface plasmon resonance, flow cytometry, Western blotting, immunocytochemistry, immunoprecipitation, and affinity chromatography. Alternatively, biochemical or cell-based assays, such as an HT-1080 cell migration assay or fluorescence-based or luminescence-based reporter assays may be employed.

In some embodiments, binding may be determined by detecting agonism or antagonism (including blocking activity in the case of ion channels and enzymes) resulting from the binding of a binding member to a target molecule, such as a ligand, receptor or ion channel. For example, the founder library, modified library or shuffled library may be screened by expressing the library in reporter cells and identifying one or more reporter cells with altered gene expression or phenotype. Suitable functional screening techniques for screening recombinant populations of binding members are well-known in the art^(16,78,79,91).

Systems suitable for the construction of libraries by cloning repertoires of genes into reporter cells have been reported^(16, 78). These systems combine expression and reporting within one cell, and typically introduce a population of antibodies selected against a pre-defined target (e.g. using phage or mammalian display).

A population of nucleic acids encoding binding members as described herein may be introduced into reporter cells to produce a library using standard techniques. Clones within the population with a binding member-directed alteration in phenotype (e.g., altered gene expression or survival) may be identified. For this phenotypic-directed selection to work there is a requirement to retain a linkage between the binding member gene present within the expressing cell (genotype) and the consequence of binding member expression (phenotype). This may be achieved for example by tethering a binding member to the cell surface, as described for antibody display or through the use of semi-solid medium to retain secreted antibodies in the vicinity of producing cells¹⁰⁵. Alternatively, binding members may be retained inside the cell. Binding members retained on the cell surface or in the surrounding medium may interact with an endogenous or exogenous receptor on the cell surface causing activation of the receptor. This in turn may cause a change in expression of a reporter gene or a change in the phenotype of the cell. As an alternative the binding member may block the receptor or ligand to reduce receptor activation. The nucleic acid encoding the binding member which causes the modified cellular behaviour may then be recovered for production or further engineering.

As an alternative to this “target-directed” approach, it is possible to introduce into a cell a “naïve” binding member population which has not been pre-selected to a particular target molecule. The cellular reporting system is used to identify members of the population with altered behaviour. Since there is no prior knowledge of the target molecule, this non-targeted approach has a particular requirement for a large repertoire, since pre-enrichment of the binding member population to the target molecule is not possible.

The “functional selection” approach may be used in other applications involving libraries in eukaryotic cells, particularly higher eukaryotes such as mammalian cells. For example, a binding member may be fused to a signalling domain such that binding to target molecule causes activation of the receptor. Suitable signalling domains include cytokine receptor domains, such as thrombopoeitin (TPO) receptor, erythropoietin (EPO) receptor, gp130, IL-2 receptor and the EGF receptor. Binding member-receptor chimaeras may be used to drive target molecule dependent gene expression or phenotypic changes in primary or stable reporter cells. This capacity may be used to identify fused binding members in the library which drive a signalling response or binders which inhibit the response¹²³⁻¹²⁵.

Combinations of recipients and linker sequences identified using the methods described herein may be used to present different donor diversity scaffold domains of the same class, for example a cysteine-rich protein with the same number of cysteine residues, or a different class.

The donor diversity scaffold domain may be replaced in the one or more identified binding members from the library, the modified library and/or the shuffled library with a substitute donor diversity scaffold domain.

The donor diversity scaffold domain and the substitute donor diversity scaffold domain may be from the same scaffold class. For example, the donor diversity scaffold domain and the substitute donor diversity scaffold domain may comprise the same scaffold and different interaction sequences. In some embodiments, the donor diversity scaffold domain may be a knottin which binds to a first target molecule and the substitute donor diversity scaffold domain may be a knottin which binds to a second target molecule. A recipient diversity scaffold domain which has been optimised for one knottin donor diversity scaffold domain as described herein may be useful for all knottin donor diversity scaffold domains. For example, a donor knottin domain within the recipient antibody variable domain of a founder binding member may be replaced with a different knottin donor domain, as exemplified below.

The donor diversity scaffold domain and the substitute donor diversity scaffold domain may be from the different scaffold classes. For example, the donor diversity scaffold and the substitute donor diversity scaffold may comprise a different donor scaffold and a different donor interaction sequence. For example, a donor knottin domain within the recipient antibody variable domain of a founder binding member may be replaced with an adhiron donor diversity scaffold domain, as exemplified below.

An additional amino acid sequence may be introduced into the fusion proteins or partner domains of the one or more identified binding members from the library and/or modified library. For example, the donor diversity scaffold domain, for example a knottin donor domain, may be engineered to introduce new binding specificities, for example by rational design, loop grafting and combinatorial library based approaches using in vitro display technologies.

The additional amino acid sequence may be a target binding sequence, such as a VEGF-A binding sequence⁹² or a Thrombopoietin (TPO) binding sequence⁵⁴.

In some embodiments, a diverse sequence may be introduced into a binding member as described herein to generate a further library for selection of improved variants. For example, diversity may be introduced into the donor or recipient diversity scaffold domain or the partner domain. Suitable diverse sequences may be introduced by oligonucleotide-directed mutagenesis or random mutagenesis²².

In some embodiments, binding mediated by a first domain selected from the donor diversity scaffold domain, recipient diversity scaffold domain and partner domain within a binding member may be used as a guide domain to direct selection from a library using the method described herein to identify modified or shuffled binding members in which a second domain also contributes to binding.

The second domain may interact with the same target molecule as the first guide domain or may interact with a second target molecule that is closely associated with it. For example, binding to the alpha sub-unit of an ion channel, such as a voltage gated sodium or potassium channel (e.g. a Nav or Kv channel) by a knottin donor diversity scaffold domain may be increased or extended by additional contacts from a partner VH domain or recipient VL domain binding either to the same alpha sub-unit or to an associated sub-unit, such as the beta subunit of an ion channel.

In a second round, the original guide domain may also be modified or replaced. For example, one of the donor diversity scaffold domain, recipient diversity scaffold domain or partner domain of a binding member may act as a first guide domain which binds to the target molecule. Having identified a second domain which contributes to binding the target molecule, a method may comprise modifying or replacing said first guide domain to produce a library of binding members comprising the second domain and a diverse first domain. This results in a final fusion protein which benefited from the availability of the original guide domain during its development as described herein but does not contain the original guide domain. This may be useful, for example if a final binding member is desired which does not contain the fusion of a donor diversity scaffold domain within a recipient diversity scaffold domain.

For example, if a knottin donor diversity scaffold domain within a binding member was used to isolate a partner antibody variable domain (e.g. a VH domain) in a first round of selection, then this partner antibody variable domain can in turn act as a guide in a second round to select for an alternative complementary variable domain (e.g. a VL domain). This complementary domain may be a recipient diversity scaffold domain with a fused donor diversity scaffold domain or may be a normal (i.e. unfused) antibody variable domain. In some embodiments, the antibody variable domain (e.g. VH domain) identified from the first cycle through use of an ion channel binding donor diversity scaffold domain may bind to an associated sub-unit of the ion channel, such as a beta chain which interacts with different alpha chains. This antibody variable domain could then guide the selection of complementary antibody variable domains or VL-donor diversity scaffold domain fusion proteins towards different alpha chains which associate with the same 3 chain.

In some embodiments, the first domain may bind to the target molecule with low to moderate affinity. For example, affinity may be less than a K_(D) 100 nM. This allows the improvements in binding due to the contribution of the second domain which may bind to the same protein or a closely associated sub-unit. Alternatively the second domain may bind to a non-interacting or weakly interacting molecule on the same cell which sequesters the second domain on the target cell facilitating its interaction with target.

In some embodiments, the target molecule may be presented on the surface of a cell in a tagged or untagged form to determine binding⁹³. For example, an integral membrane protein, such as an ion transporter or GPCR, may be expressed with a tag. A library of binding members comprising a partner domain which binds the tag (e.g. an antibody VH or VL domain) and a diverse population of donor diversity scaffold domains presented on a VL or VH recipient domain may be subjected to selection for improved binding to the tagged, cell surface expressed target protein. This may allow the identification of fusion proteins and donor diversity scaffold domains that bind the target protein outside the tag. These fusion proteins and donor diversity scaffold domains may be used in further rounds of screening, for example with diverse partner domains, to identify binding members that bind the untagged target protein.

Various suitable tags are known in the art, including, for example, MRGS(H)₆ (SEQ ID NO: 36), DYKDDDDK (SEQ ID NO: 37) (FLAG™), T7-, S-(KETAAAKFERQHMDS; SEQ ID NO: 38), poly-Arg (R₅₋₆), poly-His (H₂₋₁₀), poly-Cys (C₄) poly-Phe(F₁₁) poly-Asp(D₅₋₁₆), Strept-tag II (WSHPQFEK; SEQ ID NO: 39), c-myc (EQKLISEEDL; SEQ ID NO: 40), Influenza-HA tag (Murray, P. J. et al (1995) Anal Biochem 229, 170-9), Glu-Glu-Phe tag (Stammers, D. K. et al (1991) FEBS Lett 283, 298-302), Tag.100 (Qiagen; 12 aa tag derived from mammalian MAP kinase 2), Cruz tag 09™ (MKAEFRRQESDR; (SEQ ID NO: 41), Santa Cruz Biotechnology Inc.) and Cruz tag 22™ (MRDALDRLDRLA; (SEQ ID NO: 42), Santa Cruz Biotechnology Inc.). Known tag sequences are reviewed in Terpe (2003) Appl. Microbiol. Biotechnol. 60 523-533.

In some embodiments, a library, modified library and/or shuffled library as described herein may be subjected to selection for binding members in which the donor diversity scaffold domain and/or the recipient diversity scaffold domain adopt their native structure i.e. they are correctly folded into an active conformation.

For example, when the donor diversity scaffold domain binds to a known target molecule, the founder library, modified library and/or shuffled library as described herein may be subjected to selection for binding members bind to the target molecule. Binding members that bind to the target molecule comprise a donor diversity scaffold domain that is active and correctly folded into its native structure. This may be useful, for example, when the actual structure of the donor diversity scaffold domain is not known.

As well as affinity based selection for the correct folding of the donor diversity scaffold, selection systems such as phage display may provide additional selectivity for correct folding of the recipient since it has been shown that phage display enriches for fusions with superior structural integrity⁹⁴. When the recipient diversity scaffold domain is an immunoglobulin or a fragment or domain thereof, the library, modified library and/or shuffled library as described herein may be subjected to selection for binding members that bind to an immunoglobulin binding molecule⁹⁵. Binding members that bind to the immunoglobulin binding molecule comprise a recipient diversity scaffold domain that is active and correctly folded into its native structure despite the fusion of a donor domain. The stringency of the system could be improved further by subjecting the population of displayed elements (e.g. a phage display population) to more disruptive conditions e.g. increased temperature, as has been done previously^(94,96).

Suitable immunoglobulin binding molecules are well known in the art and include wild type and engineered forms of protein L, protein G and protein A, as well as anti-Ig antibodies and antibody fragments. In some embodiments, the immunoglobulin binding molecule may be contained in an affinity chromatography medium. Suitable affinity chromatography media are well-known in the art and include KappaSelect™, Lambda Select™, IgSelect™, and GammaBind™ (GE Healthcare).

One or more binding members identified as described above may be further tested, for example to determine the functional effect of binding to the target molecule. A method may comprise determining the effect of the one or more binding members identified from the library, the modified library and/or the shuffled library on the activity of a target molecule.

In some embodiments, the target molecule may be an ion channel and the effect of the one or more identified binding members on ion flow through the channel may be determined

Ion flux through a channel may be determined using routine electrophysiological techniques such as patch-clamping. For example, ion flux may be measured using a two electrode voltage clamp following endogenous or heterologous expression of the ion channel. In some embodiments, ion channel function may be determined using fluorescence based screens. For example, cells expressing an endogenous or heterologous ion channel, for example a voltage-gated calcium channel (Cav), may be loaded with a fluorophore, such as Fura3 or Calcium Green. Activation of the ion channel by K⁺ induced depolarisation causes a transient increase in intracellular ion concentration which can be readily measured. Suitable systems for depolarising cell membranes and recording responses are available in the art (e.g. FlexStation™; Molecular Devices Inc USA).

Ion flux through a channel may also be determined using a fluorescent protein ion sensor. Suitable fluorescent protein ion sensors are available in the art⁹⁷.

In some embodiments, the effect of the one or more identified binding members on a panel of target molecules may be determined. For example, the effect of the one or more identified binding members on ion flow through a panel of ion channels may be determined. This may be useful for example, in determining or characterising the specificity of the binding member. Alternatively the effect of the one or more identified binding members on protease activity may be determined where the target is a protease. Alternatively the effect of the one or more identified binding members on cellular signalling may be determined where the target is a receptor.

Following identification as described above, one or more identified binding members may be recovered, isolated and/or purified from a library, modified library and/or shuffled library.

In preferred embodiments, each binding member in the library may be displayed on the surface of a particle, such as a bead, ribosome, cell or virus which comprises the nucleic acid encoding the binding member.

Following selection of binding members which display the binding activity (e.g. bind the target molecule) and are displayed on bacteriophage or other library particles or molecular complexes, nucleic acid may be taken from a bacteriophage or other particle or molecular complex displaying a selected binding member.

Particles displaying the one or more identified binding members may be isolated and/or purified from the library, the modified library and/or the shuffled library. Nucleic acid encoding the one or more identified binding members may be isolated and/or purified from the particles.

The isolated nucleic acid encoding the one or more identified binding members may be amplified, cloned and/or sequenced.

Suitable methods of nucleic acid amplification, cloning and/or sequencing are well known in the art (see for example Protocols in Molecular Biology, Second Edition, Ausubel et al. eds. John Wiley & Sons).

The sequence of the isolated nucleic acid may be used in subsequent production of the binding member or fusion protein.

The one or more identified binding members or encoding nucleic acid may be synthesised. For example, the one or more binding members may be generated wholly or partly by chemical synthesis. For example, binding members, or individual donor or recipient diversity scaffold domains or fusion proteins and partner domains thereof may be synthesised using liquid or solid-phase synthesis methods; in solution; or by any combination of solid-phase, liquid phase and solution chemistry, e.g. by first completing the respective peptide portion and then, if desired and appropriate, after removal of any protecting groups being present, by introduction of the residue X by reaction of the respective carbonic or sulfonic acid or a reactive derivative thereof.

Chemical synthesis of polypeptides is well-known in the art (J. M. Stewart and J. D. Young, Solid Phase Peptide Synthesis, 2nd edition, Pierce Chemical Company, Rockford, Ill. (1984); M. Bodanzsky and A. Bodanzsky, The Practice of Peptide Synthesis, Springer Verlag, New York (1984); J. H. Jones, The Chemical Synthesis of Peptides. Oxford University Press, Oxford 1991; in Applied Biosystems 430A User's Manual, ABI Inc., Foster City, Calif.; G. A. Grant, (Ed.) Synthetic Peptides, A User's Guide. W. H. Freeman & Co., New York 1992, E. Atherton and R. C. Sheppard, Solid Phase Peptide Synthesis, A Practical Approach. IRL Press 1989 and in G. B. Fields, (Ed.) Solid-Phase Peptide Synthesis (Methods in Enzymology Vol. 289). Academic Press, New York and London 1997).

The one or more identified binding members may be recombinantly expressed from encoding nucleic acid. For example, a nucleic acid encoding a binding member may be expressed in a host cell and the expressed polypeptide isolated and/or purified from the cell culture.

Nucleic acid sequences and constructs as described above may be comprised within an expression vector. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. Preferably, the vector contains appropriate regulatory sequences to drive the expression of the nucleic acid in a host cell. Suitable regulatory sequences to drive the expression of heterologous nucleic acid coding sequences in expression systems are well-known in the art and include constitutive promoters, for example viral promoters such as CMV or SV40, and inducible promoters, such as Tet-on controlled promoters. A vector may also comprise sequences, such as origins of replication and selectable markers, which allow for its selection and replication and expression in bacterial hosts such as E. coli and/or in eukaryotic cells.

Many known techniques and protocols for expression of recombinant polypeptides in cell culture and their subsequent isolation and purification are known in the art (see for example Protocols in Molecular Biology, Second Edition, Ausubel et al. eds. John Wiley & Sons, 1992; Recombinant Gene Expression Protocols Ed R S Tuan (March 1997) Humana Press Inc).

Suitable host cells include bacteria, mammalian cells, plant cells, filamentous fungi, yeast and baculovirus systems and transgenic plants and animals. The expression of antibodies and antibody fragments in prokaryotic cells is well established in the art. A common bacterial host is E. coli.

Expression in eukaryotic cells in culture is also available to those skilled in the art as an option for production of a binding member. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney cells, NS0 mouse melanoma cells, YB2/0 rat myeloma cells, human embryonic kidney cells (e.g. HEK293 cells), human embryonic retina cells (e.g. PerC6 cells) and many others.

One or more of the identified binding members may be re-formatted. For example, a binding member comprising an immunoglobulin recipient binding domain or immunoglobulin partner domain may be re-formatted as an scFv, Fab, scFv-Fc, Fc, IgA, IgD, IgM, IgG, or half-antibody. A method may comprise isolating nucleic acid encoding the immunoglobulin domain (e.g. a VH or VL domain) from cells of a clone, amplifying the nucleic acid encoding said domain, and inserting the amplified nucleic acid into a vector to provide a vector encoding the antibody molecule. Re-formatting from an scFv format to a Fab and an IgG format is exemplified below.

In some embodiments, the donor diversity scaffold domain from the one or more identified binding members may be isolated. A method described herein may further comprise synthesising or recombinantly expressing an isolated donor diversity scaffold domain from one or more of the identified binding members from the founder library, the modified library and/or the shuffled library.

The isolated donor diversity scaffold domain may be re-formatted. For example, the donor diversity scaffold domain may be inserted into a different recipient diversity scaffold domain in a binding member described herein or may be inserted into different binding member format, such as a N or C terminal fusion

In some embodiments, the isolated donor diversity scaffold domain may be used as an independent binding molecule, free of any recipient domain or fusion partner.

A detectable or functional label or half-life extension moiety may attached to the one or more identified binding members.

A label can be any molecule that produces or can be induced to produce a signal, including but not limited to fluorescers, radiolabels, enzymes, chemiluminescers or photosensitizers. Thus, binding may be detected and/or measured by detecting fluorescence or luminescence, radioactivity, enzyme activity or light absorbance.

Suitable labels include enzymes, such as alkaline phosphatase, glucose-6-phosphate dehydrogenase (“G6PDH”), alpha-D-galactosidase, glucose oxidase, glucose amylase, carbonic anhydrase, acetylcholinesterase, lysozyme, malate dehydrogenase and peroxidase e.g. horseradish peroxidase; dyes; fluorescent labels or fluorescers, such as fluorescein and its derivatives, fluorochrome, rhodamine compounds and derivatives, GFP (GFP for “Green Fluorescent Protein”), dansyl, umbelliferone, phycoerythrin, phycocyanin, allophycocyanin, o-phthaldehyde, and fluorescamine; fluorophores such as lanthanide cryptates and chelates e.g. Europium etc (Perkin Elmer and Cis Biointernational); chemoluminescent labels or chemiluminescers, such as isoluminol, luminol and the dioxetanes; bio-luminescent labels, such as luciferase and luciferin; sensitizers; coenzymes; enzyme substrates; radiolabels including bromine77, carbon14, cobalt57, fluorine8, gallium67, gallium 68, hydrogen3 (tritium), indium111, indium 113m, iodine123m, iodine125, iodine126, iodine131, iodine133, mercury107, mercury203, phosphorous32, rhenium99m, rhenium101, rhenium105, ruthenium95, ruthenium97, ruthenium103, ruthenium105, scandium47, selenium75, sulphur35, technetium99, technetium99m, tellurium121m, tellurium122m, tellurium125m, thulium165, thulium167, thulium168 and yttrium199; particles, such as latex or carbon particles; metal sol; crystallite; liposomes; cells, etc., which may be further labelled with a dye, catalyst or other detectable group; and molecules such as biotin, digoxygenin or 5-bromodeoxyuridine; toxin moieties, such as for example a toxin moiety selected from a group of Pseudomonas exotoxin (PE or a cytotoxic fragment or mutant thereof), Diptheria toxin or a cytotoxic fragment or mutant thereof, a botulinum toxin A, B, C, D, E or F, ricin or a cytotoxic fragment thereof e.g. ricin A, abrin or a cytotoxic fragment thereof, saporin or a cytotoxic fragment thereof, pokeweed antiviral toxin or a cytotoxic fragment thereof and bryodin 1 or a cytotoxic fragment thereof.

Suitable enzymes and coenzymes are disclosed in Litman, et al., U.S. Pat. No. 4,275,149, and Boguslaski, et al., U.S. Pat. No. 4,318,980, Suitable fluorescers and chemiluminescers are disclosed in Litman, et al., U.S. Pat. No. 4,275,149. Labels further include chemical moieties, such as biotin that may be detected via binding to a specific cognate detectable moiety, e.g. labelled avidin or streptavidin. Detectable labels may be attached to antibodies of the invention using conventional chemistry known in the art.

Suitable half-life extension moieties include fusions to Fc domains or serum albumin, unstructured peptides such as XTEN⁹⁸ or PAS⁹⁹ polyethylene glycol (PEG),

A method described herein may further comprise formulating the one or more identified binding members from the library, the modified library and/or the shuffled library or the isolated donor diversity scaffold domain with a pharmaceutically acceptable excipient.

The term “pharmaceutically acceptable” as used herein pertains to compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgement, suitable for use in contact with the tissues of a subject (e.g., human) without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio. Each carrier, excipient, etc. must also be “acceptable” in the sense of being compatible with the other ingredients of the formulation. Suitable carriers, excipients, etc. can be found in standard pharmaceutical texts, for example, Remington's Pharmaceutical Sciences, 18th edition, Mack Publishing Company, Easton, Pa., 1990.

Another aspect of the invention provides a nucleic acid encoding a fusion protein or a binding member described herein.

Nucleic acid may comprise DNA or RNA and may be wholly or partially synthetic. Reference to a nucleotide sequence as set out herein encompasses a DNA molecule with the specified sequence, and encompasses a RNA molecule with the specified sequence in which U is substituted for T, unless context requires otherwise.

Another aspect of the invention provides a vector comprising a nucleic acid described herein, as well as transcription or expression cassettes comprising the nucleic acid. Vectors may be plasmids, viral e.g. ‘phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 3rd edition, Russell et al., 2001, Cold Spring Harbor Laboratory Press.

Another aspect of the invention provides a population of nucleic acids encoding a library of binding members described herein.

The nucleic acids encoding the library may be contained in expression vectors, such as phage or phagemid vectors.

Another aspect of the invention provides a population of particles comprising a library described herein and/or a population of nucleic acids encoding a library described herein.

The library may be displayed on the surface of the viral particles. Each binding member in the library may further comprise a phage coat protein to facilitate display. Each viral particle may comprise nucleic acid encoding the binding member displayed on the particle. Suitable viral particles include bacteriophage, for example filamentous bacteriophage such as M13 and Fd. Techniques for the production of phage display libraries are well known in the art.

Other aspects of the invention provide a host cell comprising a binding member, nucleic acid or vector as described herein, and a population of host cells comprising a library, population of nucleic acids and/or population of viral particles described herein.

Another aspect of the invention provides a pharmaceutical composition comprising a fusion protein or a binding member described herein and a pharmaceutically acceptable excipient.

The pharmaceutical composition may conveniently be presented in unit dosage form and may be prepared by any methods well-known in the art of pharmacy. Such methods include the step of bringing the binding member into association with a carrier which may constitute one or more accessory ingredients. In general, pharmaceutical compositions are prepared by uniformly and intimately bringing into association the active compound with liquid carriers or finely divided solid carriers or both, and then if necessary shaping the product.

Pharmaceutical compositions may be in the form of liquids, solutions, suspensions, emulsions, elixirs, syrups, tablets, lozenges, granules, powders, capsules, cachets, pills, ampoules, suppositories, pessaries, ointments, gels, pastes, creams, sprays, mists, foams, lotions, oils, boluses, electuaries, or aerosols.

A binding member or pharmaceutical composition comprising the binding member may be administered to a subject by any convenient route of administration, whether systemically/peripherally or at the site of desired action, including but not limited to, oral (e.g. by ingestion); topical (including e.g. transdermal, intranasal, ocular, buccal, and sublingual); pulmonary (e.g. by inhalation or insufflation therapy using, e.g. an aerosol, e.g. through mouth or nose); rectal; vaginal; parenteral, for example, by injection, including subcutaneous, intradermal, intramuscular, intravenous, intraarterial, intracardiac, intrathecal, intraspinal, intracapsular, subcapsular, intraorbital, intraperitoneal, intratracheal, subcuticular, intraarticular, subarachnoid, and intrasternal; by implant of a depot, for example, subcutaneously or intramuscularly.

Pharmaceutical compositions suitable for oral administration (e.g., by ingestion) may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active compound; as a powder or granules; as a solution or suspension in an aqueous or non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion; as a bolus; as an electuary; or as a paste.

Pharmaceutical compositions suitable for parenteral administration (e.g. by injection, including cutaneous, subcutaneous, intramuscular, intravenous and intradermal), include aqueous and non-aqueous isotonic, pyrogen-free, sterile injection solutions which may contain anti-oxidants, buffers, preservatives, stabilisers, bacteriostats, and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents, and liposomes or other microparticulate systems which are designed to target the compound to blood components or one or more organs. Examples of suitable isotonic vehicles for use in such formulations include Sodium Chloride Injection, Ringer's Solution, or Lactated Ringer's Injection. Typically, the concentration of the active compound in the solution is from about 1 ng/ml to about 10 μg/ml, for example, from about 10 ng/ml to about 1 μg/ml. The formulations may be presented in unit-dose or multi-dose sealed containers, for example, ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.

It will be appreciated that appropriate dosages of the binding member, can vary from patient to patient. Determining the optimal dosage will generally involve the balancing of the level of diagnostic benefit against any risk or deleterious side effects of the administration. The selected dosage level will depend on a variety of factors including, but not limited to, the route of administration, the time of administration, the rate of excretion of the imaging agent, the amount of contrast required, other drugs, compounds, and/or materials used in combination, and the age, sex, weight, condition, general health, and prior medical history of the patient. The amount of imaging agent and route of administration will ultimately be at the discretion of the physician, although generally the dosage will be to achieve concentrations of the imaging agent at a site, such as a tumour, a tissue of interest or the whole body, which allow for imaging without causing substantial harmful or deleterious side-effects.

Administration in vivo can be effected in one dose, continuously or intermittently (e.g., in divided doses at appropriate intervals). Methods of determining the most effective means and dosage of administration are well known to those of skill in the art and will vary with the formulation used for therapy, the purpose of the therapy, the target cell being treated, and the subject being treated. Single or multiple administrations can be carried out with the dose level and pattern being selected by the physician.

Binding members described herein may be used in methods of diagnosis or treatment in human or animal subjects, e.g. human. Binding members for a target molecule, such as an ion channel may be used to treat disorders associated with the target molecule.

Other aspects of the invention provide a fusion protein, binding member or composition described herein for use as a medicament; a fusion protein, binding member or composition described herein for use in the treatment of pain or autoimmune disease; and the use of a fusion protein, binding member or composition described herein in the manufacture of a medicament for the treatment of a condition associated with ion channel activity or dysfunction.

Another aspect of the invention provides a method of treatment comprising administration of a binding member or composition described herein to an individual in need thereof, optionally for the treatment of a condition associated with ion channel activity or dysfunction.

An effective amount of a binding member or composition described herein alone or in a combined therapeutic regimen with another appropriate medicament known in the art or described herein may be used to treat or reduce the severity of at least one symptom of any of a disease or disorder associated with the target molecule in a patient in need thereof, such that the severity of at least one symptom of any of the above disorders is reduced.

Other aspects and embodiments of the invention provide the aspects and embodiments described above with the term “comprising” replaced by the term “consisting of” and the aspects and embodiments described above with the term “comprising” replaced by the term “consisting essentially of”.

It is to be understood that the application discloses all combinations of any of the above aspects and embodiments described above with each other, unless the context demands otherwise. Similarly, the application discloses all combinations of the preferred and/or optional features either singly or together with any of the other aspects, unless the context demands otherwise.

Modifications of the above embodiments, further embodiments and modifications thereof will be apparent to the skilled person on reading this disclosure, and as such, these are within the scope of the present invention.

The contents of all documents, websites, databases and sequence database entries mentioned in this specification, including the amino acid and nucleotide sequences disclosed therein, are incorporated herein by reference in their entirety for all purposes.

“and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the Figures described above.

EXPERIMENTS 1. Summary

The fusion of a donor diversity scaffold (e.g. a knottin) into a recipient diversity scaffold domain (e.g. an antibody VL domain) was first exemplified herein using the trypsin inhibitor; Ecballium elaterium trypsin inhibitor-II (EETI-II). In this knottin scaffold, high affinity binding to trypsin is dependent on correct folding of the knottin. The knottin gene was cloned into either CDR1 or CDR2 of an antibody light chain recipient (demonstrated for both kappa and lambda light chains). A library of variants of the recipient was created to allow selection of those recipient variants which could accommodate the incoming donor. The donor and recipient domains were in close apposition. In the example below, the incoming donor replaced a CDR loop and the ends of the incoming donor domain were fused to the flanking antibody framework residues i.e. β strand sequences of the core structure. In some examples below, framework residues were replaced by random sequences. The binding member was thus constructed in a way that limited the length of sequence joining the donor and recipient scaffold such that few or no additional residues were added. In some cases, there were fewer residues within the junction between framework residues and the donor domain compared to what would be have been generated by a direct fusion of the secondary structural elements of donor and recipient. A library of variants was created wherein the sequence of amino acid residues at the N and C terminal boundary of the incoming domain was varied while maintaining a short linker length to limit conformational flexibility. This library was designed to identify correct fusion combinations having a fixed kappa or lambda sequence preceding the knottin with diversity introduced from a natural light chain repertoire in the region that followed the knottin insertion. Thus, for knottin insertion into CDR1, the sequence from framework 2 (FW2) to the end of the VL domain was from a natural repertoire. Likewise insertion into CDR2 was followed from framework 3 to the end of the VL domain by sequences from a natural repertoire.

The population of genes encoding the fusion of the donor and recipient diversity scaffolds was cloned into a phage display vector, phage particles displaying the fusion were recovered and the population selected on immobilized trypsin using phage display technology^(28,100). In this way, selections were carried out for retained trypsin binding and therefore correct folding of the knottin donor. As well as affinity based selection for the correct folding of the donor, phage display provides additional selectivity for correct folding of the recipient since it has been shown that phage display enriches for fusions with superior structural integrity⁹⁴. Thus, if the donor was correctly folded (and therefore capable of binding trypsin) but folding of the recipient was compromised, then display of the donor would still be compromised (possibly through degradation of the misfolded fusion). Selectivity for folding of an antibody recipient may thus be further enhanced by preselecting on protein L or protein A which have both been shown to bind to certain VL and VH domains respectively⁹⁵ with a requirement for correct folding. The stringency of the system may be improved further by subjecting the phage population to more disruptive conditions^(94,96).

A panel of light chain variants which support folding and trypsin binding of EETI-II was selected. Once a scaffold has been selected for one member of a donor diversity scaffold family (knottins, in this case) it may be used for additional members of the same donor diversity scaffold family given the shared structural constraints within the donor family. Using two of the selected recipient antibody light chain scaffolds a range of other knottins which block sodium channels⁷¹ ⁷² ⁷⁰, potassium channels^(25,73,74) and acid sensitive ion channels^(118, 122) were used as donors within the previously selected recipient antibody domain. Some of these were shown to retain blocking activity on the target ion channel, in the case of the voltage gated potassium channel Kv1.3 and the acid sensitive ion channel 1a. Thus the selected recipient domain was shown to provide a generic scaffold which can be used for multiple, distinct donors of the same class of diversity scaffold.

The two binding scaffolds were joined by short, non-flexible linkers to limit flexibility and relative movement between the domains. Given the close proximity of the fused binding scaffolds there is an opportunity for amino acids on the surface of both donor and recipient diversity scaffolds to contribute to the paratope involved in interaction with the same epitope on a single protein. Likewise the surface of partner domains of either donor or recipient diversity scaffolds may contribute to binding of target. Alternatively, both scaffolds may contact closely apposed sites on a target protein complex. Conformational flexibility carries a thermodynamic “entropic penalty” for binding interactions. For the same reason, fixing the conformational relationship between the two fused domains is advantageous, and so it is preferable to avoid flexible linker sequences to join the two domains.

A binding member comprising a donor knottin inserted into an recipient antibody variable domain were crystallised and the protein structure determined. This confirmed the correct folding of the knottin and showed that the knottin presented within the recipient antibody variable domain had the same structure as shown previously for the free knottin. The structure also confirmed correct folding of the antibody and the close apposition of the two domains. Both domains showed the expected structure and the expectation of a relative rigid conformation between the domains is supported by the crystal structure. The fusion of a cysteine-rich donor domain to an antibody recipient domain is hereafter referred to as a KnotBody™

Example 1: Construction of Knottin-VL CDR Fusion Phage Display Libraries

The phage display vector pSANG4²⁸ is a phagemid-based phage display vector which allows the facile cloning of single chain Fv (scFv) formatted antibodies. In the scFv format the VH gene and the VL gene are joined by DNA encoding a flexible linker peptide. With pSANG4 scFv genes are cloned into the Nco1 and Not 1 sites to create an in-frame fusion with the M13 leader sequence which precedes the Nco1 site and the minor coat protein encoded by gene 3 which follows the Not1 site (FIG. 1A).

A pair of synthetic scFv gene encompassing a fixed VH gene (D12) which does not interact with trypsin (FIG. 2) were synthesised (from Genscript). This D12 VH gene¹⁰¹ binds to the “TNF alpha converting enzyme” (TACE) and originates from the IGHV3-23 germ line gene (also known as DP47) which is frequently used in antibody responses¹⁰².

The VH gene is coupled to a synthetic gene encoding the N terminal part of either the IGKV1 D-39 germline gene (a member of the V kappa 1 family) or the IGLV1-36 germline gene (a member of the V lambda 1 family) up to the end of FR2 segment followed by a Not 1 restriction site.

This synthetic gene encompassing a partial VL gene will allow the insertion of a knottin gene at either the CDR1 position or the CDR2 position (FIG. 1B, C). The gene introduces a Pml1 restriction site at the end of FR1 which is used to clone a repertoire of knottins flanked at their 5′ and 3′ ends by a Pml1 and Mfe1 site respectively (FIG. 1B). The Pml site was introduced near the end of FW1 as a result of a silent mutation in the kappa gene. A Ser to Thr mutation was introduced into the FW1 region of the lambda gene to accommodate this same restriction enzyme site. The synthetic genes also introduced a Pst1 site at the end of FW2 which is used to introduce a knottin gene into the CDR2 position using restriction sites Pst1 and BspE1 at the 5′ and 3′ ends of the knottin gene (FIG. 1C). In assembling a functional VL gene, the remainder of the VL domain (from FR2 in the case of a CDR1 knottin insertion and from FW3 in the case of a CDR2 insertion) was generated by PCR from a light chain repertoire²⁸ to complete the fusions (see example 2).

These 2 synthetic gene encoding either a kappa or lambda light chain germline gene segment were amplified using primers 2561 (GGTACCTCGCGAATGCATCTAG; SEQ ID NO: 43) and 2562 (CATGCAGGCCTCTGCAGTCG; SEQ ID NO: 44). Purified PCR products were digested with Nco1 and Not1 before ligating into pSANG4 vector²⁸ cut with the same restriction enzymes. Ligations were transformed into E. coli DH5-alpha cells (Life technologies) according to manufacturer's instructions. Plasmid DNA was prepared from the sequence confirmed transformants using Macherey Nagel Midi prep kit (according to the manufacturer's instructions). These vector were named as “pIONTAS1_KB_Kappa” and “pIONTAS1_KB_lambda” and the sequences from the the Nco1 site to the Not1 site are shown in FIGS. 2A and 2B respectively.

To demonstrate the potential of fusing a folded knottin donor with an antibody recipient, Ecballium elaterium trypsin inhibitor-II (EETI-II), a plant derived knottin with trypsin inhibitory activity was used. EETI-II has free N and C termini and trypsin binding is driven by the constrained sequence “PRIL” present in loop 1 (FIG. 3A)¹⁰³. The high affinity for trypsin in the correctly folded state¹⁰⁴ offers the potential to identify donor knottins which retain trypsin binding despite being cloned into a recipient VL domain. The construction of a phage display library where variable fusion sequence have been introduced between the donor and recipient will allow selection of functional knottin-CDR-VL combinations from the libraries based on trypsin binding.

This knottin donor diversity scaffold was fused into either the CDR1 or CDR2 position of either kappa or lambda VL domains. This was done by replacing residues within the incoming knottin donor and the VL recipient with random amino acids in a way that limited the number of added amino acids between the 2 domains or even reduced them (FIG. 3B). We also introduced a repertoire of light chain fragments encompassing the segment of the VL gene following the point of insertion (example 2).

Insertion of Knottins into CDR1

A synthetic EETI-II Knottin donor gene was made and was used to replace the CDR1 positions in the light chain recipient by creating a PCR fragment with Pml1 and Mfe sites (which were introduced by PCR) and cloning into pIONTAS1_KB_Kappa and pIONTAS1_KB_lambda. Framework and CDR designations are as described by the International Immunogenetics Information System (IMGT)²⁹. The PCR primers also introduced variable amino acid sequences between the restriction sties and the knottin gene. The PCR primers Pml1-5EETa and Pml1-5EETb each introduced 3 variable codons encoded by the sequence VNS (V=AC or G, N=ACG or T, S=C or G) immediately after the Pml restriction site. The “VNS” degenerate codons encode 16 amino acids (excluding cysteine, tyrosine, tryptophan, phenylalanine and stop codons) at each randomised position from 24 codon combinations.

Pml1-5EETa (SEQ ID NO: 45) GTGT VNS VNS VNS TGC CCG CGT ATC CTG ATG CGT TGC Pml1-5EETb (SEQ ID NO: 46) GTGT gga VNS VNS VNS TGC CCG CGT ATC CTG ATG CGT TGC

The 5′ end of these sequences represents the site created by Pml1, a restriction enzyme recognising the sequence “CACGTG” (SEQ NO: 47) to create a blunt end. To facilitate blunt end cloning of the PCR product primers with 5′ phosphorylated termini were synthesised.

The introduction of the PCR products at this point has the effect of replacing the last 3 residues of FW1 and the first residue of EETI-II (which forms part of a β sheet within the knottin) with 3 randomised residues. The net effect is the loss of an amino acid in the join between the framework and donor. Primer Pml1-5EETb encodes an additional Gly residue which restores the number of residues between them (FIG. 3B).

At the 3′ end of the knottin gene the antisense primers 1-5EETMfe (encoding an Mfe1 site) was used to amplify EETI-II. The Mfe1 site used for cloning encodes the 2 and 3rd amino acids of FW2 (Asn-Trp). This primer also retained the terminal glycine found in EETI-II and introduced 2 random amino acids encoded by VNS or VNC codons followed by an Mfe1 site.

The consequence of this strategy for introducing a knottin donor gene is to remove the first amino acid of FW2 and join the 2 domains using 2 randomised amino acids (FIG. 3B) with a net addition of 1 amino acid between the donor and framework.

1-5EETMfe (SEQ ID NO: 48) GTCAGTCCAATTGNBSNBCCCGCA GAAGCCGTTCGGACCGCA

Sequences encoding the Mfe1 site are underlined. As an example, the constructs resulting from amplification with Pml1-5EETa and 1-5EETMfe followed by cloning into either pIONTAS1_KB_lambda and pIONTAS1_KB_Kappa are shown in FIGS. 3C and 3D respectively.

Insertion of Knottins into CDR2

The knottin donor was introduced into the CDR2 positions of pIONTAS1_KB_lambda and pIONTAS1_KB_Kappa by creating a PCR fragment with Pst1 and BspE1 sites which was introduced by PCR. It was possible to introduce a Pst1 site into the first two residues of the CDR2 region of the IGKV1 D-39 V kappa gene (within the Ala-Ala sequence). For convenience in cloning the same restriction site and encoded residues were introduced at the end of framework 2 of the V lambda germline gene IGLV1-36. In the lambda germline it was also possible to introduce a BspE1 site into the 4th and 5th residues of the FW3 region encoding Ser-Gly) (FIG. 3E). Again this restriction site and encoded amino acids were also introduced into the V kappa gene (FIG. 3F).

The PCR primers PST1-5EETa was used to amplify EETI-II and introduces 2 Ala codons encompassing a Pst1 site for cloning (underlined) followed by 2 variable codons encoded by the sequence GNS (encoding Val, Ala, Asp or Gly) and VNS (encoding 16 amino acids within 24 codons) immediately after the Pst1 restriction site and preceding the knottin sequence. The VNS codon replaces the first amino acid of EETI-II with the net effect of adding 3 amino acids (2 Ala residues and one of Val, Asp, Ala or Gly) between the donor and recipient framework (FIG. 3B).

PST1-5EETa (SEQ ID NO: 49) ATC TAT GCT GCA GNS VNS TGC CCG CGT ATC CTG ATG CGT TGC

At the 3′ end of the incoming knottin donor, The PCR primers −5EETBse was used to amplify EETI-II. This introduces a BspE1 site for cloning. The PCR product introduces the knottin donor followed by the glycine which naturally follows the last cysteine of EETI-II (FIG. 3A).

This is followed by 2 randomised amino acids which adjoin to the 4th and 5th amino acids of FW3 after cloning. This has the effect of replacing the first three residues of FW3 with two randomised amino acids and retains the terminal glycine of the knottin (FIG. 3B). The net effect is the loss of an amino acid between donor and framework 3.

1-5EETBse (SEQ ID NO: 50) GTCAGAGACTCCGGASNBSNBCCCGCA GAAGCCGTTCGGACCGCA

As an example, the constructs resulting from amplification with PST1-5EETa and 1-5EETBse followed by cloning into either pIONTAS1_KB_Kappa or pIONTAS1_KB_lambda are shown in FIGS. 3E and 3F respectively.

Example 2. Introduction of Additional Diversity into the Recipient VL Domain

In humans there are 2 different classes of antibody light chain, (kappa or lambda) which are encoded by approximately 50 and 40 germline genes respectively. These can each be arranged into families on the basis of sequence homology with 7 different kappa families (V kappa 1-Vkappa 7) and 11 different lambda families (V lambda 1-Vlambda 11). Examination of the germline sequences²⁹ permits the design of sense strand primers (“forward primers”) specific for different germline VL gene families.

In the course of antibody maturation individual VL germline genes are recombined with approximately 5 different kappa or lambda-specific J segments at their 3′ end. It is possible therefore to design a small number of forward and reverse primers to enable the amplification of re-arranged VL genes from human donors for construction of antibody phage display libraries as described for example in Schofield et al (2007)²⁸. In this example we demonstrate how a similar approach can be used to amplify fragments of VL genes beginning at either FW2 or FW3 to the end of the VL gene. This allows introduction of diversity into recipient VL domains which have donor insertions in either CDR1 or CDR2. This approach therefore allows the introduction of additional diversity into a recipient domain downstream of the inserted donor.

Amplification of VL Repertoires Beginning at Framework 2.

Sequences of the families of V kappa and V lambda germline genes were aligned (data from IMGT²⁹) and PCR primers were designed to allow amplification of VL repertoires from the beginning at Framework 2. Lambda-specific primers (“Mfelam”) capable of amplifying from the FW2 region of V lambda 1, 2 and 3 were designed. These primers were also designed to introduce an Mfe1 site (underlined) which is compatible and in frame with the Mfe1 site introduced at the end of the knottin gene described above. In a similar way, kappa-specific primers (“Mfekap”) capable of amplifying from the FW2 region of V kappa 1, 2 and 3, were designed.

Mfelam 1 (SEQ ID NO: 51) GTGTGGAGGTGGC AAT TGG TAC CAG CAG CTC CCA GG Mfelam 2 (SEQ ID NO: 52) GTGTGGAGGTGGC AAT TGG TAC CAA CAG CAC CCA GG Mfelam 3 (SEQ ID NO: 53) GTGTGGAGGTGGC AAT TGG TAC CAG CAG AAG CCA GG Mfekap 1 (SEQ ID NO: 54) GTGTGGAGGTGGC AAT TGG TAT CAG CAG AAA CCA GG Mfekap 2 (SEQ ID NO: 55) GTGTGGAGGTGGC AAT TGG TAC CTG CAG AAG CCA GG Mfekap 3 (SEQ ID NO: 56) GTGTGGAGGTGGC AAT TGG TAC CAG CAG AAA CCT GG

These sense strand forward primers were used to amplify a repertoire of VL genes from a library which had previously been pre-cloned into the Nhe1/Not1 site of a phage display vector (as shown in FIG. 1). The introduced Mfe1 site is underlined and the sequence of Mfelam1 and Mfekap1 are represented at the beginning of FW2 in FIGS. 3E and 3F respectively. For kappa light chains a single reverse primer (JKFOR_Thr2) was designed which straddled the original cloning junction encompassing Not1 including homology to the phagemid vector as well as the beginning of the cloned J kappa segment. For lambda light chains, a single primer (JLFORQP2) was designed which hybridised within the phagemid plasmid sequence, across the Not1 cloning site and into the beginning of J lambda segment.

JLFORQP2 (SEQ ID NO: 57) TGAGATGAGTTTTTGTTCTGCGGCCGCGGGCTGACCTAG JKFOR_Thr2 (SEQ ID NO: 58) TGAGATGAGTTTTTGTTCTGCGGCCGCGGTACGTTTGAT

Amplification with the paired kappa or lambda forward and reverse primers generated a PCR product encoding:

(SEQ ID NO: 59) Mfe1-FW2-CDR2-FW3-CDR3-FW4-Not1.

The primers introduce an Mfe1 site at the 5 end and a Not 1 site at the 3′ end, compatible and in frame with the knottin PCR products described above and the phagemid display vector shown in FIG. 1. This will allow the amplification of libraries of VL genes which can be fused to constructs encoding a knottin insertions at CDR1 by using restriction enzymes Mfe1 and Not 1. This introduces additional CDR2 and CDR3 diversity into the recipient VL domain. JLFORQP2 and JKFOR_Thr2 are anti-sense reverse primers and the reverse complementary encoding sense strand is represented in FIGS. 3C, 3D, 3E and 3F respectively as the FW4-Not1 region in the final construct.

Amplification of VL Repertoires Beginning at Framework 3

By a similar approach, sense strand forward primers were designed which hybridised with the beginning of FW3 of lambda VL genes (“Bsplam” primers) or kappa (“BspKap” primers) while introducing a BspE1 restriction site compatible with the BspE1 site introduced into the knottin constructs described above. The introduced BspE1 site is underlined and the sequence of Bsplam1a and BspKap1a are represented at the beginning of FW3 in FIGS. 3E and 3F respectively.

Bsplam1a (SEQ ID NO: 60) ATCTATGCTGCAGGAGGTGGC TCC GGA GTC TCT GAC CGA TTC TCT GG Bsplam1e (SEQ ID NO: 61) ATCTATGCTGCAGGAGGTGGC TCC GGA GTC cCT GAC CGA TTC TCT GG Bsplam2 (SEQ ID NO: 62) ATCTATGCTGCAGGAGGTGGC TCC GGA TCC AAG TCT GGC AAC ACG GG Bsplam3 (SEQ ID NO: 63) ATCTATGCTGCAGGAGGTGGC TCC GGA ATC CCT GAG CGA TTC TCT GG BspKap1a (SEQ ID NO: 64) ATCTATGCTGCAGGAGGTGGC TCC GGA GTC CCA TCA AGG TTC AGT GG BspKap1b (SEQ ID NO: 65) ATCTATGCTGCAGGAGGTGGC TCC GGA GTC CCA TCA AGG TTC AGC GG BspKap2 (SEQ ID NO: 66) ATCTATGCTGCAGGAGGTGGC TCC GGA GTC CCA GAC AGG TTC AGT GG BspKap3 (SEQ ID NO: 67) ATCTATGCTGCAGGAGGTGGC TCC GGA ATC CCA GcC AGG TTC AGT GG

When lambda-specific forward primers were used with the lambda-specific reverse primer (JLFORQP2) or when kappa-specific forward primers were used with the kappa-specific reverse primers ((JKFOR_Thr2) then a PCR product encoding the sequence below was produced.

BspE1-FW3-CDR3-FW4-Not1

This allowed the amplification of libraries of VL genes which can be fused to constructs with a knottin gene insertions at CDR2 by using restriction enzymes BspE1 and Not 1. This introduced additional CDR3 diversity into the recipient VL domain.

Using standard molecular biology techniques, constructs (as depicted in FIG. 3) were created using gene fragments created as described above. These were cloned into pIONTAS1_KB_Kappa” and “pIONTAS1_KB_lambda and were electroporated into E. coli TG1 cells and KnotBody library sizes of 3.5-6.6×10⁸ were constructed using standard molecular biology techniques e.g. Schofield et al 2007²⁸.

Example 3: Isolation and Characterisation of Functional Knottin-Antibody Fusion Clones from the KnotBody Libraries Using Phage Display Technology

Phage display technology facilitates isolation of binders to any given target from large combinatorial repertoires based on proteins and peptides that can be presented on the surface of filamentous phage¹⁰⁵. In order to isolate functional knottin-antibody fusions from the KnotBody libraries multiple rounds of phage display selections on trypsin (cognate antigen for the EETI-II donor) were carried out. Phage particles were prepared by rescuing KnotBody libraries using trypsin cleavable helper phage^(102,106) Two rounds of phage display selections were performed on 10 μg/ml biotinylated trypsin immobilised on Streptavidin (for Round-1) or Neutravidin (for round-2) coated Maxisorp™ immunotubes (Thermo Scientific, Cat No. 444202). In both rounds of selection, phage libraries were pre-incubated with streptavidin coated paramagnetic beads (Thermo Fisher Scientific, Cat. No. 11205D). These beads were removed prior to the addition the phage to the antigen tubes to limit the streptavidin binders entering the selection. Polyclonal phage preparations from round-2 selection outputs were analysed for trypsin binding using a time resolved fluorescence (TRF) binding assay. In this assay, polyclonal phage binding to biotinylated trypsin immobilised on neutravidin (Thermo Fisher Scientific, Cat. No 31000) coated Maxisorp™ plates (Thermo Scientific, Cat. No. 437111) was detected using a mouse anti-M13 antibody (GE Healthcare, Cat. No. 27-9420-01) followed by europium conjugated anti-mouse antibody (Perkin Elmer, Cat. No. AD0124).

Polyclonal phage from both EETI-II libraries showed good binding to trypsin in this assay, demonstrating the success of phage display selection in enriching KnotBodies that bind to trypsin (FIG. 4). To investigate whether presentation of EETI-II by standard phage display approach would work the EETI-II gene was cloned into a phagemid display vector²⁸ where it was directly fused to the N-terminus of gene III. Surprisingly EETI-II presented on phage, as a direct N-terminus gene-III fusion showed no detectable binding to trypsin while selected polyclonal phage from both EETI-II CDR fusion libraries showed trypsin binding. Importantly this result demonstrates that indirect presentation of knottins on phage via antibody CDR loops (which is fused to gene-III) could be a superior alternative to direct knottin-gene III fusion.

In order to identify monoclonal KnotBodies with desired binding characteristics, 94 individual clones derived from 2 rounds of selection using EETI-II CDR1 or CDR2 libraries were picked into 96 well culture plates and phage were prepared from each clone. Methods for screening by monoclonal phage ELISA are known in the art¹⁰⁷. The phage supernatant from each clone was tested for binding to biotinylated trypsin immobilised on neutravidin coated Maxisorp™ plates using a TRF assay (as described above). A representative example of the monoclonal binding assay is shown in FIG. 5. Clones with >3× binding signal above background were picked as positive clones and sequenced to identify unique clones. 14 and 42 unique binders were identified from EETI-II CDR1 and EETI-II CDR2 libraries respectively (Table 1). 18 binders from the EETI-II CDR2 library and 3 binders from the EETI-II-CDR1 library were chosen for further characterisation based on their VL and linker sequence diversity. These binders were assigned new clone identification numbers (Table 2).

The trypsin binding capability of EETI-II is attributed to proline (Pro3) and arginine (Arg4) residues present in its loop1 (GCPRILMRC; SEQ ID NO: 68). In order to show that the selected KnotBodies bind to trypsin via a fully folded EETI-II displayed as a VL CDR fusion (ie not via other CDR loops or C or N-terminal fusion sequences), trypsin-binding residues (Pro3 and Arg4) of EETI-II were substituted for glycine (Gly) and alanine (Ala) respectively. Monoclonal phage was prepared from the 21 selected clones and their mutant equivalents. The phage supernatant from each clone was tested for binding to biotinylated trypsin (immobilised on streptavidin coated Maxisorp™ plates) using a TRF binding assay (as described above). Replacement of the trypsin-binding residues of EETI-II abolished the ability of all KnotBody clones to bind to trypsin (Table 3). In order to confirm that the loss of trypsin binding was not due to reduced expression or display of pIII fusion phage was prepared from 11 (randomly chosen) KnotBodies and their GlyAla variants were analysed on a western blot using an antibody which recognises the product of gene 3 (pIII) using an anti-pIII antibody (MoBiTec GmbH) (FIG. 6). No significant difference in the amount of pIII fusion displayed was observed between any KnotBody clone and its GlyAla substituted variant. These data confirm that the trypsin binding functionality of these Knotody clones is resultant of correct display of the knottin donor (EETI-II) as VL CDR fusion and confirms that trypsin binding is directed by the EETI-II knottin donor.

Example 4. Crystal Structure of KnotBodies Selected from EETI-II CDR2 Library

In order to generate crystal structures of KnotBodies, 18 clones isolated from the EETI-II CDR2 library (Table 3) were reformatted as Fabs. In this format the VH (from D1A12) gene is fused to a human IgG1 heavy chain constant domain 1(CH1) and the recipient VL chains containing the EETI-II are fused to a light chain constant domain (CL). Methods for expressing antibodies by transient expression in mammalian cells are well known to those skilled in the art¹⁰⁸. In order to enable the expression of these KnotBody Fabs in mammalian cells, VL chains encoding the knottin were PCR amplified using primers LLINK2 (CTCTGGCGGTGGCGCTAGC; SEQ ID NO: 69) and NotMycSeq (GGCCCCATTCAGATCCTCTTCTGAGATGAG; SEQ ID NO: 70). Amplified VL genes were digested with restriction enzymes NheI and NotI and ligated into the pINT12 vector (encoding D1A12 heavy chain) pre-digested with the same enzymes. The pINT12 vector (FIG. 7) has a dual promoter expression cassette in which the heavy chain expression is controlled by the cytomegalovirus (CMV) promoter and the light chain expression is driven by elongation factor-1 alpha (EF1-alpha) promoter.

In order to express the KnotBody Fabs in HEK-293F cells, transfection quality DNA was prepared using Plasmid Plus Kit (Qiagen, Cat. No 12945). 24 μg of plasmid DNA was incubated with 48 μg of polyethylenimine (PEI) in 2 mls of Freestyle 293 expression medium (Thermo Fisher Scientific, Cat. No. 12338-018) for 15 minutes. After the incubation, DNA:PEI mix was added to 20 mls of HEK-293F cells (Thermo Fisher Scientific, Cat. No. R790-07) seeded at a density of 1×10⁶ cells/ml in 125 ml tissue culture flasks. Culture flasks were incubated at 37° C. for 5 days (with 5% CO₂, 75% humidity and shaking at 800 rpm). All transfection except KB_A03 successfully expressed KnotBody Fabs. Expressed proteins were purified from the cell culture supernatants using CaptureSelect IgG-CH1 affinity matrix (Life Technologies, Cat. No. 194320005) according to manufacturer's instructions. Purified proteins were dialysed against 2×PBS overnight and dialysed samples were visualised on a SDS-PAGE gel using Quick Coomassie staining (Generon, product reference, GEN-QC-STAIN). A representative example is shown in FIG. 8A. The monomeric state of the purified KnotBody Fabs was confirmed by analytical size exclusion chromatography using a Superdex200 2.4 mL column (GE HealthCare, 17-1089-01) and Akta Purifier system (GE Healthcare). All KnotBody Fabs showed chromatographic profiles corresponding to monomeric Fab species (FIG. 8B).

In order to confirm the trypsin binding ability of KnotBodies in Fab format, purified proteins were tested for binding to trypsin using a TRF binding assay. In this assay binding of KnotBody Fabs (at 10 μg/ml, 2.4 μg/ml and 0.5 μg/ml) to biotinylated trypsin (immobilised on streptavidin coated Maxisorp™ plates) was detected using a mouse anti-CH1 antibody followed by a europium conjugated anti-mouse antibody (Table 4). 7 KnotBodies (KB_A04, KB_A01, KB_A05, KB_A07, KB_A08, KB_A10, KB_A12) were chosen for crystallisation. DNA prepared for these clones were transfected into 500 ml HEK-293F cells as described before. Expressed proteins were purified using CaptureSelect IgG-CH1 affinity matrix. Purified proteins were initially subjected to crystallisation trials using commercially available crystallisation screens (Supplier). Well-defined crystals were readily obtained for KB_A12 and KB_A05. Crystallisation conditions for these two clones were further optimised and the following condition was used to generate diffraction quality crystals: 0.1 M MES pH=6.5, 25% w/v PEG MME 2000 and 0.1M Tris.Cl, 50% PEG MME, 10 mM NiCl2. Complete diffraction data was collected on these crystals at 100K under liquid nitrogen stream. Collected data was processed using PROTEUM2 software (Bruker). The crystal structure of the knottin-fused Fab antibodies was resolved using molecular replacement technique¹⁰⁹ using Phenix software v 1.8.1-1168¹¹⁰. Resulting structure was further refined using Phenix.Refine¹¹¹. The templates for molecular replacement were downloaded from PDB website (www.rcsb.org/pdb, PDB codes: 3G04 for light chain and 3QOS for heavy chain). KnotBody KB_A12 and KB_A05 sequences are given below. An additional AlaSer sequence is introduced at the N terminus of the light chain from the Nhe1 site used in cloning.

KB_A12_Heavy chain (SEQ ID NO: 71) EVQLVESGGGLVRPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSA ISGSGGSTYYADSVKGRFTISRDNTKNSLYLQMTSLRADDTAFYYCVDFG PGYGTGWFDYWGPGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLV KDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQ TYICNVNHKPSNTKVDKKVEPKSC KB_A12_Light chain (SEQ ID NO: 72) QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIY AAGR CPRILMRCKQDSDCLAGCVCGPNGFCG ANSGVSDRFSAAKSGTSAS LAINGLRSEDEADYYCAAWDDSLNGYVFGTGTKLTVLGQPAAAPSVTLFP PSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN NKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS KB_A05 Heavy chain (SEQ ID NO: 73) Evqlvesggglvrpggslrlscaasgftfssyamswvrqapgkglewvsa isgsggstyyadsvkgrftisrdntknslylqmtslraddtafyycvdfg pgygtgwfdywgpgtlvtvssastkgpsvfplapsskstsggtaalgclv kdyfpepvtvswnsgaltsgvhtfpavlqssglyslssvvtvpssslgtq tyicnvnhkpsntkvdkkvepksc KB_A05 Light chain (SEQ ID NO: 74) QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIY AADK CPRILMRCKQDSDCLAGCVCGPNGFCG GGSGIPERFSGSKSGTSAS LAISGLRSEDEADYYCATWDDNLNGVVFGGGTKLTVLGQPAAAPSVTLFP PSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSN NKYAASSYLSLTPEQWKSHKSYSCQVTHEGSTVEKTVAPTECS

For both the data sets, molecular replacement solutions were successful indicating that the overall fold of the knottin-fused Fab did not change. Upon fine refinement (FIG. 9), it was evident that the knottin-fused Fab antibody was correctly folded and the disulfide pattern of the knottin part within the antibody was exactly same as that of EETI-II (FIG. 10A), which has been reported earlier (PDB ID: 1H9I). In addition the relative arrangement of the EETI-II part from the Fab indicates that the CDR regions of the heavy chain are accessible for potential binding to other antigen. Lack of electron density for VH CDR3 residues (FIG. 10B) further strengthens this possibility. PDB coordinates

Example 5: Demonstration of Bi-Specific Binding Capability of the KnotBody Format

The concept of bi-specific antibodies that can bind to two different target antigens is increasingly popular in drug development¹¹². Conventional bi-specific antibodies are typically composed of two VH-VL pairs with different specificities (i.e. two distinct VH-VL domain pairs that each recognise a different antigen). In this example, we describe the development of a bi-specific KnotBody format. In this format, the binding determinants required for recognition of two distinct antigens are incorporated within a single VH-VL pair. This was achieved by using the VH to mediate one binding specificity and the knottin-VL fusion to mediate the other binding specificity.

Two well-characterised KnotBody trypsin binders (KB_A07 and KB_A12, see example 3) were used here as model scaffolds for engineering the bi-specific functionality. These KnotBodies contains a common heavy chain gene from D1A12 antibody (anti-TACE antibody)¹⁰¹ and two distinct light chain genes displaying EETI-II as a CDR2 fusion. The trypsin binding capability of these KnotBodies is completely attributed to the VL domain displaying EETI-II. Therefore, these fusions of recipient VL:donor knottin were recombined with a large repertoire of naïve VH genes²⁸ to generate a library from which novel molecules with additional VH mediated binding specificities can be isolated.

In a previous work Schofield and colleagues described the cloning of VH and VL gene repertoires into an intermediate antibody library²⁸. This intermediate antibody library was used to create a functional antibody phage display library (“McCafferty library” and the “Iontas antibody library”²⁸). The same repertoire of VH genes was used in this example for the construction of the KnotBody heavy chain shuffled library. This was achieved by PCR amplifying VH genes from the Iontas antibody library bacterial stocks using primers M13 leaderseq (AAATTATTATTCGCAATTCCTTTGGTTGTTCCT; SEQ ID NO: 75) and HLINK3 (CTGAACCGCCTCCACCACTCGA; SEQ ID NO: 76). Amplified VH genes were digested with restriction enzymes Nco1 and Xho1 and ligated into the pIONTAS1_KB vector (encoding KB_A017 and KB_A12 light chain) pre-digested with the same enzymes. The ligation product was purified using MiniElute PCR purification kit (Qiagen, Cat. No. 28004) and electroporated into E. coli TG1 cells (Lucigen, Cat. No. 60502-2). Phage was rescued from this library as described in example 3¹⁰⁷

In order to identify bi-specific KnotBodies with novel binding specificities mediated by the VH domain, two rounds of phage display selections were carried out against 5 different antigens (human-cMET-Fc fusion, mouse-FGFR4-CD4 fusion, human-GAS6-CD4 fusion, human-urokinase type plasminogen activator and β-galactosidase). cMET-Fc, FGFR4-CD4, GAS6-CD4 and urokinase type plasminogen activator (UPA) were immobilised directly on Maxisorp™ immunotubes. biotinylated β-galactosidase (bio-B-gal) was indirectly immobilised on Maxisorp™ immunotubes coated with streptavidin (round-1) or neutravidin (round-2). Phage selections were performed as described in example 3. Polyclonal phage prepared from the round-2 selection outputs were tested in a TRF binding assay (as described in example 3) against all 5 antigens, trypsin and the immobilisation partners (streptavidin and neutravidin). All polyclonal phage populations showed specific binding to trypsin and the antigen they were selected against but not other antigens (FIG. 11). For example, the binding signals obtained for polyclonal phage prepared from cMET-Fc selection were specific to trypsin and cMET. This result demonstrates the success of phage display selection in enriching bi-specific binders. In order to identify monoclonal binders, 48 individual clones were picked from each selection output and monoclonal phage was prepared from each clone (as described in example 3). Monoclonal phage supernatant from each clone was tested for binding to trypsin and the antigen used for selection (representative example of the screen is shown in FIG. 12). Any clone that showed a binding signal above 15000 fluorescent units on both trypsin and the selection antigen was considered to be a bi-specific binder. Large panels of bi-specific binders were identified from each selection output (Table 5).

Example 6: Demonstration of Bi-Epitopic Binding Capability of the KnotBody Format

In the previous example we demonstrated the capability of a knottin scaffold to bind to two different antigens via distinct paratopes, one located on the recipient-donor fusion (ie VL-knottin) and the other on the VH. The same principle can be applied to target two different epitopes within a single target antigen where VH directed binding could be used for increasing the affinity and/or specificity of the original interaction (mediated by the knottin). By this approach, the additional binding properties of the VH domain can be utilised to improve the potency or specificity of knottin based drugs. Alternatively, such additional contribution to binding can also be directed by VL CDR3 using the approach described in example 2. In this example, we demonstrate bi-epitopic binding capability of the knottin scaffolds by improving the affinity of anti-trypsin KnotBodies (KB_A07 and KB_A12, see example 3) by introducing novel heavy chain partners that bind independently to trypsin. KB_A07 and KB_A12 contain a common VH domain (D12) that binds to the dis-cys domain of tumour necrosis factor-α converting enzyme¹⁰¹ and a VL domain that binds to trypsin via EETI-II (displayed at the CDR2 position). In order to identify novel heavy chain partners that bind to a distinct epitope on trypsin (and improve the overall affinity), a phage display library was generated by replacing the D1A12 VH of these two KnotBodies with a large repertoire of naïve heavy chains (see example 5). Affinity improved clones were isolated from this “chain-shuffled” library by performing three rounds of phage display selections on trypsin under stringent conditions that favour preferential enrichment of high affinity clones. The stringency of selection was controlled by using decreasing amounts of trypsin in each round of selection (FIG. 13). The optimum antigen concentration for each round was determined empirically by selecting the KnotBodies against a range of antigen concentrations and comparing the phage output numbers with a no-antigen control. Round 1 selection was carried out by panning on antigen immobilised on Maxisorb immunotubes as described in example 5. The population from round 2 selections, carried out at 20 nM antigen concentration, was chose for a further round 3^(rd) round.

KnotBody genes from round 2 (20 nM selection) and round 3 (2 nM and 0.2 nM selections) outputs were PCR amplified using primers M13 leaderseq (See example 4) and NotMycSeq (GGCCCCATTCAGATCCTCTTCTGAGATGAG; SEQ ID NO: 70). PCR products were digested with NcoI and NotI restriction enzymes and cloned into the pBIOCAM5-3F vector via NcoI and NotI. The pBIOCAM5-3F allows the expression of antibodies as soluble scFv-Fc fusion proteins in mammalian cells¹¹³, hence facilitating the screening for affinity improved KnotBody binders. 352 clones (resulting from the pBIOCAM5-3F cloning) were picked into 4×96 well cultural plates. Transfection quality plasmid DNA was prepared for each clone using the Plasmid Plus 96 kit (Qiagen. Cat. No. 16181). 600 ng of each plasmid DNA was incubated with 1.44 μg of polyethylenimine (PEI) in Freestyle HEK-293F expression medium (Thermo Fisher Scientific, Cat. No. R790-07) for 15 minutes before adding to 96-well deep well plates containing 500 μl/well of exponentially growing HEK-293F cells at a density of 1×10⁶ cells/ml. Plates were sealed with gas permeable seals and incubated at 37° C. for 5 days (with 5% CO₂, 75% humidity and shaking at 800 rpm). Cell culture supernatant containing KnotBody-Fc fusion proteins was used for screening to identify affinity-improved clones.

The binding signal observed for a particular clone in the primary binding screen (e.g. TRF binding assay using monoclonal phage supernatant) is a combined function of affinity and expression. Since protein expression can vary significantly from clone to clone, ranking KnotBodies by their binding signal in the primary screen might not necessarily correlate with their affinities. Therefore two expression-independent screening assays were used to rank the KnotBody clones by affinity and compare to them with parent clones. There were (i) TRF capture assay and (ii) Biacore off-rate screen. In the “TRF capture assay” (FIG. 14), KnotBody-scFv-Fcs were captured on Maxisorp™ plates coated with an anti-Fc antibody (Abcam, Cat. No. ab113636) and the binding of biotinylated trypsin to KnotBodies was detected using streptavidin conjugated europium (Perkin Elmer, Cat. No 1244-360). To normalise for differences in KnotBody (scFv-Fc) expression, limiting amounts of anti-Fc antibody was coated on each well. For example, a well coated with 50 μl of anti-Fc antibody at a concentration of 2.5 μg/ml (32 nM) has a maximum (theoretical) capture capacity of 1.6 fmoles of scFv-Fc. However, the average scFv-Fc expression in HEK-293F cells using pBIOCAM5-3F vector system is 10-20 mg/l (83-166 nM) and 50 μl of the raw supernatant used for the assay will have 4.1-8.2 fmoles of scFv-Fc on average, hence saturating the immobilised anti-Fc antibody coated on each well. The majority of the KnotBodies selected from VH shuffled libraries showed higher binding signal against trypsin than the parent clones (representative example is shown in FIG. 15). 90 KnotBodies that gave the highest binding signal in the capture assay were cherry-picked and tested in the “Biacore off-rate screen”. In the “Biacore off-rate screen” the dissociation constants (off-rates) of the KnotBody clones were analysed by surface plasmon resonance (using Biacore T100 from GE healthcare). The dissociation constant of an antibody-antigen interaction is concentration independent and therefore normalisation for differential expression was not required. In this assay, a CM5 sensor chip (GE healthcare, Cat. No. BR-1005-30) was coupled to approximately 3000 resonance units (RU) of anti-Fc antibody (human antibody capture kit, GE healthcare, Cat. No. BR-1008-39). Approximately 1200-1600 RU of KnotBody-scfv-Fc was captured on the anti-FC antibody. The trypsin solution (400 nM) was injected for 60 seconds. The dissociation phase of each KnotBody trypsin interaction was monitored for 300 seconds. After each cycle, the chip surface was regenerated by injecting 3M MgCl₂. The dissociation constant (kd) for each KnotBody was determined using 1:1 Langmuir binding model (using Biacore T100 evaluation software). Clones that showed 1.5 fold slower dissociation constants than their parent clones were considered to have improved affinity (Table 6). Overall, 22 affinity improved clones were identified using capture assay and the off-rate screen. A representative example of a clone that showed improved off-rate compared to the parent clones is shown in FIG. 16. This improvement in the affinity of the KB_A07 and KB_A12 clones by heavy chain shuffling demonstrates the bi-epitopic binding capability of the KnotBody format.

Example 7: Cloning, Expression and Purification of Various Cysteine-Rich Toxin Donors within CDR3 of an Antibody VL Recipient Scaffold

Aberrations in normal ion channel functioning are involved in the pathogenesis of number of disease conditions including pain disorders and auto-immunity. For example defects in the function of the voltage gated sodium channel 1.7 (Nav1.7) play a major role in both hypersensitivity and insensitivity to pain. The voltage gated potassium channel 1.3 (Kv1.3) is implicated in T-cell mediated autoimmunity. The acid sensitive ion channel 1a (ASIC1a) is involved in the sensing and processing of pain signals. Thus, specific modulators of ion channels offer a great therapeutic potential in the treatment of pain and various autoimmune disorders. Number of naturally occurring knottins function as ion channel blocking toxins. As discussed earlier, the only knottin based drug (Ziconotide) approved for therapy is an N-type voltage gated calcium channel blocker derived from the venom of the conus snail.

This example describes the fusion of alternative cysteine-rich toxin donors into the previously selected recipient VL scaffolds to create KnotBodies directed towards ion channels (Table 7). This was achieved by cloning three Nav1.7 blockers, three Kv1.3 blockers and three acid sensing ion channel 1a (ASIC 1a) blocker into the CDR2 position of two KnotBodies KB_A07 and KB_A12 (i.e. replacing the EETI-II gene at that position, as described in example 3). Genes encoding all toxins (Table 8) were synthesised as gene fragments (from Integrated DNA Technologies). Each toxin gene was PCR amplified using primer sets that encodes junctional sequences specific to KB_A07 and KB_A12 (Table 9). An additional variant of Ssm6a (Ssm6s_GGS) incorporating linker sequences (GGS at the N-terminus and SGG at the C-terminus) was also PCR amplified. In this example, the KnotBody constructs were formatted as IgG molecules where the VH gene is fused to IgG1 heavy chain constant domains (CH1-CH2-CH3) and the recipient VL chains containing the new donor toxins are fused to a light chain constant domain (CL). In order to express these KnotBody constructs in mammalian cells, PCR fragments were cloned into the PstI and BspEI sites of KB_A12 and KB_A07 VL chain (encoded by the pINT3-hg1 vector, FIG. 17). The pINT3-hg1 vector has a dual promoter expression cassette in which the heavy chain expression is controlled by the cytomegalovirus (CMV) promoter and the light chain expression is driven by elongation factor-1 alpha (EF1-alpha) promoter.

In order to express the KnotBodies in mammalian cells, transfection quality DNA was prepared using Plasmid Plus Kit (Qiagen, Cat. No 12945). 60 μg of plasmid DNA was incubated with 120 μg of polyethylenimine (PEI) in 5 mls Freestyle 293 expression medium (Thermo Fisher Scientific, Cat. No. 12338-018) for 15 minutes before adding to 50 mls of HEK-293F cells (Thermo Fisher Scientific, Cat. No. R790-07) seeded at a density of 1×10⁶ cells/ml in a 250 ml tissue culture flask. Culture flasks were incubated at 37° C. for 5 days (with 5% CO₂, 75% humidity and shaking at 800 rpm). Expressed proteins were purified from the cell culture supernatants using Protein-A affinity chromatography (Protein-A sepharose from Generon, Cat. No. PC-A5). Purified proteins were dialysed against 2×PBS and dialysed samples were visualised on a SDS-PAGE gel using Quick Coomassie staining (Generon, product reference, GEN-QC-STAIN). A representative example is shown in FIG. 18. The average expression levels (7.7 mg/l) obtained for these fusion molecules in HEK293F cells were comparable to that of conventional antibodies produced using the same vector system (4 mg/l). Protein yield after purification is given in Table 10. In the case of mambalgin fusions, DNA was transfected into CHO-Expi cells as described in example 11 and these yields are given.

In summary, results described in this example illustrate that (i) ion channel blocking toxins can be expressed as donors within a fusion with VL domain recipients (e.g. KnotBodies); (ii) knottin scaffolds other than EETI-II can be formatted as KnotBodies or in other words, the KnotBody format is capable of functional presentation of multiple knottins (ii) These fusion molecules can be expressed and purified efficiently as IgGs.

Example 8: Functional Validation of Kv1.3 and ASIC1a Blockers Expressed as KnotBodies

Knottin Kv 1.3 and ASIC1a blockers expressed in the KnotBody format (see examples 6 and 7) were tested for their ability to inhibit the function of the ion channel targets. The effect of these KnotBodies on cells expressing the human Kv 1.3 (huKv1.3) or human ASIC1a channels was assessed using QPatch automated whole-cell patch-clamp electrophysiology (Sophion).

For the testing of Kv1.3, CHO cells stably expressing huKv1.3 (Charles River Lab) were analysed using the QPatch electrophysiology assay in the presence or absence of the KnotBody fusions or the antibody isotypes (negative) controls. Internal and external physiological solutions were freshly prepared prior to the assay. The extracellular solution contained 145 mM NaCl, 4 mM KCl, 2 mM CaCl2), 1 mM MgCl2, 10 mM HEPES and 10 mM glucose; the pH was adjusted to 7.4 with NaOH; the osmolarity was ˜305 mOsm/L. The intracellular solution contained: KF (120 mM), KCl (20 mM), HEPES (10 mM) and EGTA (10 mM); the pH was adjusted to 7.2 with KOH; osmolarity was ˜320 mOsm/L.

A series of depolarising voltage (channel activating) pulses were used to monitor the channel currents upon adding control (extracellular solution) to define a control baseline of current activity. To these measured control currents ascending concentrations of KnotBodies or parental antibody isotypes (control molecules) were applied to the external bathing solution (2.2 μM-0.5 nM, diluted in the extracellular solution). KnotBodies KB_A12 and KB_A07 (EETI-II fusions) were used as isotype controls for the testing. From a holding voltage of −80 mV a depolarising, activating voltage pulses of +30 or +80 mV were applied for 300 ms, every 5 or 10 seconds. The elicited, maximum outward current values measured during the activating step for each solution exchange period were averaged (n=3-8 for each concentration applied) and plotted as concentration-response curves. From these concentration-response curves (see example plots in FIG. 21) IC₅₀ values were calculated (summarised in Table 11A). KB_A07_Shk, KB_A12_Shk and KB_A07_Kaliotoxin (KTX) fusion proteins all inhibited the huKv1.3 currents in a concentration dependent manner with IC₅₀s of approximately 20 nM, 40 nM and 900 nM respectively. In comparison, the parental KnotBodies, KB_A12, KB_A07, Moka-1 on both recipient scaffolds and Kaliotoxin on KB_12 showed no significant reduction in measured current (˜IC₅₀ extrapolated to >10 μM), which were similar in magnitude to the reduction in current recordings made in control external solution additions over the same time period (four applications each of four minutes duration, 16 minutes in total).

To functionally assess the ASIC1a KnotBodies, HEK293 cells stably expressing huASIC1a (Sophion) were analysed using a QPatch electrophysiology assay in the presence or absence of the KnotBody fusions (using KB_A12 as a recipient and replacing the original EETI-II donor as shown in Table 8). The “parental” KB_A12 with the EETI-II donor was used as a negative control. Internal and external physiological solutions were freshly prepared prior to the assay. The extracellular solution contained 145 mM NaCl, 4 mM KCl, 2 mM CaCl2), 1 mM MgCl2, 10 mM HEPES and 10 mM glucose; the pH was adjusted to 7.4 with NaOH (for control, resting-state pH) or 6.0 with MES (for acidic, activating-state pH); the osmolarity was ˜305 mOsm/L. The intracellular solution contained: CsF (140 mM), HEPES (10 mM), NaCl (10 mM) and EGTA/CsOH (1 mM/5 mM); the pH was adjusted to 7.3 with CsOH; osmolarity was ˜320 mOsm/L.

Throughout ASIC1a recordings the recording cell membrane voltage was clamped at −60 mV. ASIC1a baseline control currents were elicited by applying external solution at activating pH 6.0 for 3000 ms. This activating pH application was repeated three further times, giving four baseline control ASIC1a currents. The recording cells were then pre-incubated with KnotBody diluted in external solution at pH 7.4 for 15-20 minutes. Following this KnotBody pre-incubation the same concentration of KnotBody in external solution at activating pH 6.0 was applied to the cell recording.

At the end of each KnotBody incubation and subsequent ASIC1a current activation, a full blocking concentration (300 nM) of the ASIC1a knottin inhibitor PcTx1 (positive control) was perfused onto the cell recording and a final activating pH 6.0 external solution was applied. The percentage remaining signal between activating pH 6.0 baseline (i.e. pre-KnotBody) and post-KnotBody incubation was determined. From these figures (see Table 11B), it can be seen that KnotBodies KB_A12_PcTx1 and KB_A07_PcTx1 were active, reducing the ASIC1a current to 4.8% which is the same level as seen with positive control application of PcTx1 (1.9-3.8%). KB_A12_Mba-1 and KB_A12_Mba-2 showed the same level of current as the KB_A12 negative control or the “buffer only” control.

The data above illustrates the potential to fuse knottins which have therapeutic potential (i.e. ion channel blockers) into a recipient antibody scaffold which was originally selected for fusing to a different knottin (i.e. EETI-II).

Example 9: Functional Fusion of Non-Knottin Donors to Recipient VL Domains of Antibodies

This example describes the fusion of a non-knottin donor diversity scaffold domain to a recipient VL domain. Non-knottin donor diversity scaffold domains that lack disulfide bonds could be valuable alternative to knottins. As discussed before a diverse number of protein scaffolds have been engineered for novel binding specificities^(38, 114). Here we use a small protein scaffold (approx. 100 amino acids) called an Adhiron, which is based on consensus sequence of plant-derived phystostatins (protein inhibitors of cysteine proteases). Adhirons shows high thermal stability and express well in E. coli ⁴⁶. They are structurally distinct from knottins and do not contain any cysteine residues.

As donors we chose two engineered adhirons, which bind the lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) to replace EETI-II at the VL CDR2 position of VL chains (recipients) from KB_A07 and KB_A12 (example 3). Sequences for these adhirons (referred to as Ad-LOX1-A and Ad-LOX1-B, Table 12) were obtained from patent WO2014125290 A1 and were as synthesised as gene fragments to enable cloning (Table 13). Each adhiron gene was amplified using primer sets specific for KB_A07 and KB_A12 (Table 14) using PCR. PCR products were cloned into the Pst1 and BspE1 sites of the pIONTAS1_KB phage display vector (FIG. 1) encoding KB_A12 and KB_A07 genes.

In order to test the functionality of these adhiron-antibody fusions, monoclonal phage prepared from each clone was assessed for binding to LOX-1 in a TRF binding assay. In this assay, LOX-1 (immobilised directly on Maxisorp™ plates) binding of phage displaying adhiron-antibody fusion was detected using a mouse anti-M13 antibody (GE Healthcare) followed by europium conjugated anti-mouse antibody (Perkin Elmer). Parent KnotBodies KB_A07 and KB_A12 (EETI-II fusions) were included as controls. Proteins cMET-FC, GAS6-CD4, FGFR4-CD4, UPA and B-gal were used to examine non-specific binding. The result from this assay (FIG. 19) shows that all adhiron antibody fusions specifically bind to the LOX-1. The parent clones, KB_A07 and KB_A12, showed no detectable binding to the LOX-1 confirming that LOX-1 binding shown by the adhiron-antibody fusion molecules is mediated by the adhirons (displayed in the VL CDR2 loops of these antibodies).

In this example, we have demonstrated that non-knottin donor diversity scaffolds can be inserted into a recipient VL scaffolds to create alternative KnotBody formats.

Example 10. Identification of Loop Regions for Insertion of Donor Sequence or Introduction of Diversity in Donor, Recipient or Partner Sequences

The method described in example 1 to insert a donor knottin sequence into a recipient Ig domain can be applied to the identification of sequences which join secondary structural elements of other scaffolds as potential sites for donor insertion.

For Ig domains, the framework and CDR designations are as described by the International Immunogenetics Information System (IMGT) database. According to the IMGT v3.1.10, numbering CDR1, CDR2 and CDR3 sequences occupy amino acid positions 27-38, 56-65 and 105-117 respectively (for all VH and VL sequences). FW1, FW2 and FW3 residues occupy 1-26, 39-55 and 66-104. At the end of the re-arranged V gene FW4 is encode by 6 J segments for VH, 5 J segments for V kappa and 7 J segments for V lambda (in each case encoding 11, 10 and 10 amino acids respectively for each FW4).

In one approach, an individual CDR may be removed in its entirety and replaced by an incoming donor sequence which is flanked by linkers of no more than 4 amino acid residues at each of the N and C termini. In another approach, some CDR residues may be retained but the total number of residues, including CDR residues, which link the incoming donor diversity scaffold to the scaffold of the recipient Ig diversity scaffold may not exceed 4 amino acids at each terminus (to retain close proximity between donor and recipient). In yet another approach, variation framework residues at the boundary of the junction may be removed from the donor or recipient scaffolds and replaced by randomized codons encompassing some or all amino acids. The overall linkage at N and C termini (excluding the replacement framework residues) shall not exceed 4 amino acids.

The gene or population of genes encoding the fusion designed in this way can be created using methods known to those skilled in the art (also exemplified in examples herein). The gene may be made entirely as a synthetic gene or created from gene fragments arising from restriction digestion or PCR and incorporated into the final fusion gene by PCR assembly or ligation. Gene fragments and/or PCR products can be joined ligation independent cloning, Gibson cloning, NEB Builder (NEB) or other methods known to those skilled in the art. The resultant genes can then be cloned into an appropriate expression vector as discussed above.

The approach above takes advantage of the IMGT database as a curated source of sequence information on Ig domains. The same approach described above may be used to cover all domains with a known structure. References to the pdb files which describe individual protein structures can be found in original citations (e.g. in references cited herein or references therein). The RCSB protein data bank (“pdb server”) represents a portal to structural information on biological macromolecules such as protein domains. Other sources for accessing structural data, including PDBSUM have been summarised¹¹⁵. The data associated with such files also allows identification of secondary structure elements by viewing 3D structures or by using software such as DSSB or websites running such software¹¹⁶. Using this approach, secondary structural representations upon a primary amino acid sequence may be derived. Alternatively, structural model or direct sequence alignment between a desired recipient domain and orthologues or paralogues with known structure could be used to identify secondary structure elements. Alternatively, algorithms for prediction of secondary structure elements could be used

The structure of the 10^(th) type III cell adhesion domain of fibronectin¹¹⁷ may be used to exemplify an approach to identify potential sites within a candidate recipient domain for insertion donor sequences. The same approach can be used to identify regions which could be amenable to diversification in both donor, recipient and partner chains.

As an example, the pdb file describing the 10^(th) type III cell adhesion domain of fibronectin (FnIII-10) is “1FNA”. Using the pdb file (e.g. by viewing on the pdb server) a representation of the linear amino acid sequence of FnIII-10 annotated with secondary structural elements can be generated (FIG. 20A). The regions which form beta strands are underlined and the residues which join them on the upper face of the domain are shown in lower case. In a further example the gp2⁵¹ has a structure represented by pdb file 2WMN. A linear representation of the secondary structure elements is represented in FIG. 20B. Residues joining beta strands or a beta strand-alpha helix junction are underlined.

These “joining sequence” represented in lower case represent regions which could be replaced partially or in their entirety while maintaining a short linker between donor and recipient. As discussed above, framework residues may be removed at the boundary of the junction of the recipient scaffolds and replaced by randomized codons encompassing some or all amino acids. These same residues represented in FIG. 20 also represent possible sites amenable to introduction of diversity as described above.

Gp2 is a small protein domain of 64 amino acids with N and C termini which are in proximity and so this molecule also represents an ideal candidate for a donor domain. In addition it has been shown that the N and C termini of Gp2 can be truncated without affecting folding of the domain allowing fusion in close proximity to a recipient domain.

Example 11. Selected Linkers are Required for Functional Expression of Knottin Donor Domains within an Antibody Recipient Domain

In contrast to peptide fusion approaches employed by others which utilise long flexible linkers for joining domains, the KnotBody format uses short non-flexible linker sequences to limit the relative movement between the donor and recipient domains. This is an important aspect of the invention. In order to identify suitable linkers permitting correct folding of 2 fused structural domains we have adopted the approach of making a library with variation in the linker. In example 3 and example 12 we have described the isolation of several functional KnotBody molecules with selected, short, non-flexible linker sequences joining the EETI-II (donor domain) with framework residues of VL or VH domains (recipient).

To demonstrate the importance of linker selection the selected short linkers of two well characterised KnotBodies, KB_A07 and KB_A12, were replaced with the long flexible N- and C terminal linkers typically used by others^(11, 13, 15) (See table 15). The performance of these linker variants were then compared to that of the original KnotBodies with short non-flexible linkers selected from the library using a trypsin binding assay.

In this example, the KnotBody variants were formatted as Fabs where the VH gene is fused to the heavy chain constant domain-1 (CH1) and the VL chains containing KnotBody linker variants are fused to the light chain constant domain (CL). The VL genes encoding the knottin and linker sequences were synthesised as gene fragments (Integrated DNA Technologies, See table 16). In order to express the KnotBody linker variants in mammalian cells, these gene fragments were cloned into the pINT12 expression vector (encoding D1A12 heavy chain) using Nhe1 and Not1 restriction sites.

The pINT12 vector (FIG. 7) has a dual promoter expression cassette in which the heavy chain expression is controlled by the cytomegalovirus (CMV) promoter and the light chain expression is driven by elongation factor-1 alpha (EF1-alpha) promoter.

In order to express the KnotBodies in mammalian cells transfection quality DNA was prepared using NucleoBond Xtra Midi plus kit (Macherey Nagel, Cat. No. 740412.50). 25 μg of plasmid DNA was incubated with 80 μl of ExpiFectamine CHO transfection reagent (Thermo Fisher scientific, Cat. No. A29129) in 2 mls of OptiPRO Serum Free Medium (Thermo Fisher Scientific, Cat. No. 12309050) for 5 minutes before adding to 25 mls of Expi-CHO cells (Thermo Fisher Scientific, Cat. No. A29133) seeded at a density of 6×10⁶ cells/ml in a 125 ml tissue culture flask. Culture flasks were incubated at 37° C. for 7 days (with 5% CO2, 75% humidity and shaking at 800 rpm) with a single feed 18-22 hour post transfection. The feed included 6 mls of ExpiCHO feed containing 150 μl of enhancer solution. Expressed proteins were purified from cell culture supernatants using CaptureSelect IgG-CH1 affinity matrix (Thermo Fisher Scientific, Cat. No. 194320005) according to manufacturer's instructions. Purified proteins were dialysed against 2×PBS overnight. Protein concentrations were determined using absorbance at 280 nm and theoretical extinction co-efficient.

In order to compare the performance of the KnotBodies with short and long linkers, purified Fabs were tested for binding to trypsin by ELISA. In this assay binding of KnotBody Fabs (at 0.5 μg/ml) to biotinylated trypsin (5 μg/ml immobilised on streptavidin coated Maxisorp™ plates) was detected using a mouse anti-CH1 antibody (Hybridoma Reagent Laboratories) followed by a europium conjugated anti-mouse antibody (Perkin Elmer, Cat. No. AD0124). The trypsin binding capability of KB_A07 and KB_A12 KnotBodies were greatly diminished when the short selected linkers were replaced with long and flexible linkers (FIG. 22). This example clearly demonstrates the importance of short non-flexible linkers for generating optimal donor-recipient fusions.

Example 12. Introduction of Diversity into Donor Domain by Randomising Donor Interaction Domains

Examples 5 and 6 showed the use of VH shuffling to generate sequence diversity within the binding partner domain of a KnotBody to select for variants with additional specificity or improved binding kinetics. In both examples, diversification was performed on the partner domain (VH domain) and the original binding specificity of the donor domain (knottin) was unchanged. In examples 2 and 3 we illustrated the introduction of diversity into the VL recipient domain. In this example, we demonstrate a different approach to introduce sequence diversity in the donor domain of a KnotBody: replacing the existing loops in the donor knottin with random amino acid sequences. The trypsin binding capability of the KnotBodies described in this patent was conferred by the loop 1 sequences (PRILMR) of the knottin-EETI-II. Here we replaced the loop 1 sequences of KB_A12 KnotBody with randomised sequences of varying lengths (6, 8, 9 and 10 residues) to increase diversity as well as potential binding surface. The resulting loop libraries were then used to generate cMET and β-galactosidase binding specificities using phage display technology. In order to create large phage display libraries with high proportion of loop substituted variants an oligonucleotide-directed mutagenesis approach using Kunkel mutagenesis (Kunkel, T. A., et al. Meth. Enzymol. 154, 367-382 (1987), and selective rolling circle amplification Huovinen, T. et al. PLoS ONE 7, e31817 (2012) was used. Oligonucleotides used encoded VNS codons (V=A/C/G, N=A/GIC/T and S=G/C) that encode 16 amino acids (excludes cystiene, tyrosine, tryptophan, phenylalanine and stop codons) at a given position. The mutagenic oligonucleotides used, representing the antisense strand (where B=TGC), are given in Table 18.

The template DNA encoding KB_A12 (in the phage display vector, pSANG4²⁸) was purified as uracil containing single stranded DNA (dU-ss DNA) from phage rescued from E. coli CJ236 (a dut⁻/ung⁻ strain). The antisense oligonucleotides described above were annealed to the dU-ssDNA template and extended using T7 polymerase to produce the complimentary strand. Newly synthesised heteroduplex DNA was subjected to rolling circle amplification using random hexamers (Thermo Fisher Scientific, Cat. No. S0142) and Phi29 polymerase (Thermo Fisher Scientific, Cat. No. EP0091) to selectively amplify the mutant strand. The product of rolling circle amplification was cut to plasmid size units using NotI restriction endonuclease (New England Bio labs, Cat. No. R3189S) and re-circularised by self-ligation.

The ligation product was purified using MiniElute PCR purification kit (Qiagen, Cat. No. 28004) and electroporated into E. coli TG1 cells (Lucigen, Cat. No. 60502-2). Methods for kunkel mutagenesis and selective rolling circle amplification are known to those skilled in the art. Phage was rescued from this library as described in example 3.

In order to isolate KnotBody loop binders with novel specificities, two rounds of phage display selections were carried out against human cMET and β-galactosidase immobilised directly on directly on Maxisorp™ immunotubes. Phage selections were performed as described in example 3. KnotBody VL genes from round 2 outputs were PCR amplified using primers LLINK2 (SEQ ID NO: 69) and NotMycSeq (SEQ ID NO: 70). Amplified KnotBody VL genes were digested with NheI and NotI and ligated into the pSANG4 vector (encoding D1A12 heavy chain) pre-digested with the same enzymes. The ligation products were transformed into E. coli TG1 cells. In order to identify monoclonal binders, 47 individual clones were picked from each transformation and monoclonal phage was prepared from each clone (as described in example 3). Monoclonal phage supernatant from each clone was tested for binding to the antigen used for selection using a TRF binding assay. 30/47 clones tested bound c-Met and 37/47 clones bound β-galactosidase (FIG. 23). Binders from this assay were sequenced using the primer LMB3 (CAGGAAACAGCTATGACC: SEQ ID NO: 77). 23 unique sequences were identified for cMET (Table 17A) and 11 unique sequences identified for β-galactosidase (Table 17B). Although the natural length of loop 1 of EETI-II is 6 residues, all novel binders isolated from the loop library possessed a loop size of either 8 or 9 amino acids.

This example clearly demonstrates that additional diversity can be introduced on to a KnotBody by substituting the existing knottin loops with random sequences of varying lengths. This loop substitution/randomisation approach can be used to acquire a new specificity (as described in examples 5) or fine tune the existing specificity or affinity mediated by the donor or recipient domain.

Example 13: Generation of Bi-Specific KnotBodies that can Cross the Blood Brain Barrier (BBB) Via Receptor Mediated Transcytosis (RMT)

Crossing the blood-brain barrier (BBB) constitutes a major challenge for the delivery of peptides and antibodies into the brain for the treatment of neurological disorders (e.g. chronic pain, Alzheimers disease, multiple sclerosis, epilepsy). For example, approximately 0.1% of circulating antibodies cross into the brain (Zuchero, Y. J. Y. et al. Neuron 89 p 70-82 (2015). Alternatively, therapeutics such as Ziconotide (a knottin from Conus snail venom) are administrated using intrusive and expensive intrathecal injection procedures. Hence, the development of effective strategies to deliver molecules across the BBB has been a long-standing goal for the pharmaceutical industry (Niewoehner, J. et al. Neuron 81, 49-60 (2014). In recent years, the main focus of this research has been the generation of molecules capable of shuttling drug moieties across the BBB by taking advantage of endogenous nutrient transport systems for example receptor mediated transcytosis (RMT). This is generally achieved using a bi-specific format that includes a “molecular shuttle” and an active the drug moiety. For example, Yu and co-workers have reported a conventional bi-specific antibody which transports an anti-Beta secretase-1 (BACE) inhibitor across the BBB using a “molecular shuttle” that binds transferrin receptor (TFR) (Yu, Y. J. et al. Sci Transl Med 3, 84ra44-84ra44 (2011)). This bi-specific antibody format consists of 2 distinct VH: VL pairs (ie 2 different Fab arms) which individually recognise either TFR or BACE. In example 5, we have demonstrated the generation of bi-specific KnotBodies where the binding determinants required for recognition of two distinct antigens are incorporated within a single VH-VL pair. In this example, we describe the generation of bi-specific KnotBodies in the same format where VH domains selected against TFR shuttle their partner Knottin-VL fusion domains across the BBB via RMT.

Example 5 describes VH shuffled libraries composed of trypsin binding KnotBody light chains (KB_A07 and KB_A12) partnered with a repertoire of VH genes isolated from non-immunised donors. Anti-TFR bi-specific KnotBodies were selected from these libraries using 2-3 rounds of phage display selections on TFR antigen directly immobilised on Maxisorp™ immunotubes. Round 1 selection was carried out on mouse TFR followed by round 2 selection on rat TFR. In another case, 3 rounds of selections were carried on mouse TFR-mouse TFR-rat TFR (representing antigens used in rounds 1, 2 and 3 respectively). Alternatively, selections were carried out on mouse TFR-human TFR-mouse TFR. 736 individual clones were then picked from the final round of selections and monoclonal phage was prepared from each clone (as described in example 3). Monoclonal phage supernatant from each clone was tested for binding to rat TFR by ELISA (a representative example of the screen is shown in FIG. 24). 376 clones that showed a binding signal above 10,000 fluorescent units were picked and sequenced to identify unique clones. Based on the analysis of VH CDR sequences, 65 unique binders were identified.

In order to enable the expression of these TFR binding KnotBodies as Fabs, VH genes were PCR amplified using primers M13 leaderseq (see example 4) and HLINK3 (CTGAACCGCCTCCACCACTCGA: SEQ ID 76). Amplified VH genes were digested with restriction enzymes Nco1 and Xho1 and ligated into the pINT12 vector (encoding KB_A07 or KB_A12 light chain) pre-digested with the same enzymes. The pINT12 vector (FIG. 7) has a dual promoter expression cassette in which the heavy chain expression is controlled by the cytomegalovirus (CMV) promoter and the light chain expression is driven by elongation factor-1 alpha (EF1-alpha) promoter. In order to express these KnotBody Fabs in mammalian cells transfection quality DNA was prepared using Plasmid Plus Kit (Qiagen, Cat. No 12945). 15 μg of plasmid DNA was incubated with 48 μl of ExpiFectamine CHO transfection reagent (Thermo Fisher scientific, Cat. No. A29129) in 1 ml of OptiPRO Serum Free Medium (Thermo Fisher Scientific, Cat. No. 12309050) for 5 minutes before adding to 15 mls of Expi-CHO cells (Thermo Fisher Scientific, Cat. No. A29133) seeded at a density of 6×106 cells/ml in a 125 ml tissue culture flask. Culture flasks were incubated at 37° C. for 7 days (with 5% CO2, 75% humidity and shaking at 800 rpm) with a single feed 18-22 hour post transfection. The feed included 3.6 mls of ExpiCHO feed supplemented with 90 μl of enhancer solution. Expressed proteins were purified from cell culture supernatants using CaptureSelect IgG-CH1 affinity resin (Thermo Fisher Scientific, Cat. No. 194320005) according to manufacturer's instructions. Purified proteins were dialysed against 2×PBS overnight. Protein concentrations were determined using absorbance at 280 nm and theoretical extinction co-efficient.

In order to assess the ability of these KnotBodies to cross the BBB, a ready to use in vitro rat BBB model system (Pharmacocell, Cat. No. RBT-24H) was used. In this model system rat brain endothelial cells were co-cultured with pericytes and astrocytes to mimic the in vivo BBB anatomy (Nakagawa, S. et al. (2009) Neurochem. Int. 54, 253-263). The in vitro rat BBB kit was cultured according to manufacturer's instructions and transendothelial electrical resistance (TEER) was measured prior to testing of KnotBodies to verify the integrity of the barrier. All wells had a TEER measurement of >200 Ω/cm2. 21 KnotBody Fabs were tested for their ability to cross the in vitro BBB model system. OX-26, a well characterised anti-rat TFR antibody that has been shown to cross BBB in vivo models (albeit at low levels) was used a positive control (Jefferies, W. A., Brandon, M. R., Williams, A. F. & Hunt, S. V. Immunology 54, 333-341 (1985), Moos, T. & Morgan, E. H. Journal of Neurochemistry 79, 119-129 (2001)). The parent KnotBody KB_A12 was used as a negative control. Both positive and negative controls were expressed and purified in the Fab format as described above. For the assay, test KnotBodies were prepared as 7 oligoclonal mixes each containing 3 different Fab antibodies at a final concentration of 2 μM in the assay buffer (DMEM-F12 with 15 mM HEPES, 0.5% BSA, 500 nM hydrocortisone and 10 μg/ml Na—F). The positive and negative controls were prepared at 1 μM in the same assay buffer. These Fab oligoclonal mixes and controls were added to the upper chamber (referred as blood side) of the co-culture kit and incubated at 37° C. for 6 hours (with 5% CO2 and 75% humidity). The concentration of transcytosed Fabs in the lower chamber (referred as brain side) was determined using a sandwich ELISA. In this ELISA, Fabs were captured using an anti-CH1 antibody (provided by Hybridoma Reagent Laboratories) and detected using rabbit anti-human IgG Kappa light chain antibody (Abcam, Cat. No. ab195576) or anti-human IgG Lambda light chain antibody (Abcam, Cat. No. ab124719) followed by europium conjugated anti-rabbit antibody (Perkin Elmer, Cat. No. AD0105). Oligoclonal KnotBody Fab mixes in wells C2, C3, and C4 showed significant levels of transcytosis compared to the negative controls (see table 19). VH sequences of KnotBodies present in these wells are shown in Table 20. This example demonstrates the use of a VH domain specific to a receptor (i.e. TFR) involved in macromolecule transport into the brain to deliver a Knottin-VL fusion with a different specificity (trypsin binding in this example). The same approach could be used to generate functional Knottin-VL fusions that can modulate the activity of targets that are expressed in the brain (e.g. ion channels inside central nervous system, proteases such as BACE). The level of transcytosis observed can be further improved by optimising the VH domain specificity and/or affinity using methods known to those skilled in the art.

Example 14: Comparison of a KnotBody and a Bovine Antibody with a Natural “Ultra Long VH CDR3 (Cow ULVC-Ab) Presenting a Knottin Insertion (EETI-II Cow ULVC-Ab)

Bovine antibodies have been described with natural ultra-long VH CDR3s containing a solvent exposed double stranded antiparallel sheet approximately 20 A° in length (referred to as the stalk) presenting a folded domain stabilised by 3 disulfide bonds (referred to as the Knob)¹⁰. Zhang and colleagues have shown that the Knob domains of these cow ultra-long VH CDR3 antibodies (ULVC-Ab) can be replaced with other folded proteins such as erythropoietin¹³. In this example, we assess the ability of a cow ULVC-Ab to present functional knottin fusions on phage in comparison with a KnotBody. In order to create a Knottin-cow UCLA fusion, the sequence between the first and the last cysteines of the knob region of the Cow ULVC-Ab BLV1H12 was replaced with the sequence of the trypsin binding knottin; EETI-II (EETI-II cow ULVC-Ab). A gene fragment encoding this construct was synthesised (Integrated DNA technologies, Table 21). This synthetic gene was cloned into the phage display vector pSANG4 using restriction sites NcoI and NotI and transformed into E. coli TG1 cells. The performance of the Knottin-Cow ULVC-Ab fusion was then compared to the KnotBody KB_A12 (which presents the knottin EETI-II as VL CDR2 fusion) using a trypsin binding assay. In this assay, the binding of phage rescued from EETI-II cow ULVC-Ab and KB_A12 to trypsin was detected using an anti-M13 antibody followed by europium conjugated anti-mouse antibody (as described in example 3). The KnotBody KB_A12 exhibited superior binding to trypsin compared to the EET-II-Cow ULVC-Ab fusion (FIG. 25).

Example 15. Functional Display of KnotBodies on the Surface of Mammalian Cells

In examples 1, 2, 3, 5, 6, 9, 12 and 13 phage display technology was employed to enable the display of KnotBodies or derivative libraries and their selection. As an alternative to phage display, display of binders has been carried out on bacterial, yeast and mammalian cells. In these methods, it is possible to use flow sorting to measure binding and expression of the binding molecule as well as binding to target. Thus, the display of libraries of KnotBodies on the surface of cells e.g. mammalian cells will enable the screening and selection of tens of millions of clones by fluorescent activated cell sorting (FACS) for increased affinity to the target and expression level in their final scFv-Fc, Fab or IgG format. Methods for constructing and using display libraries in other systems are known to those skilled in the art.

Libraries of KnotBodies, (e.g. within recipient, donor, linkers or partner domains) may also be used for direct functional screening for altered cellular phenotypes in mammalian cells as described above. Thus, libraries of displayed or secreted KnotBodies in mammalian cells may provide an efficient route to discovery of the drug candidates or lead molecules. Methods for the construction of libraries in mammalian cells by nuclease directed integration are provided, for example, in WO2015166272 and the references cited therein, all of which are incorporated herein by reference. In this example, we demonstrate that it is possible to display a membrane anchored KnotBody scFv-Fc on the surface of mammalian cells. This was achieved by cloning the KnotBodies KB_A07 and KB_A12 scFv into the targeting vector pD6 (FIG. 26), performing nuclease mediated gene integration into HEK293 cells followed by flow cytometry analysis. The flow cytometry results showed that expression was achieved when staining with labelled anti-Fc and that the displayed knottin was correctly folded because staining was also achieved with fluorophore-labelled trypsin.

The KnotBody genes KB_A07 and KB_A12 were prepared for insertion into a mammalian display vector in a way that can be modified to create linker libraries. 3 fragments were produced and assembled by PCR using overlap at the boundaries to drive the association. In an N terminal to C terminal direction fragment 1 consists of a PCR product encoding the N terminal part of the scFv gene up to the donor insertion point (encoding in this case Nco1 site, entire VH, scFv linker, FW1, CDR1, FW2, overlap). Fragment 2 consists of overlap, incoming knottin donor, overlap. Fragment 3 consists of overlap, FW3, CDR3, FW4 Not 1 site). Description of potential linker sizes and sites are given below. The position of the overlap can be chosen but will typically be homologous to the antibody framework or the donor coding region (allowing diversity to be introduced in one or other fragment internally to the overlap. In examples below the fragments include between 18 to 27 nucleotide homology overlaps to enable their assembly and amplification by PCR. The final scFv product, encompassing the donor is generated by a 3-fragment assembly. This 3-fragment assembly approach is described in detail using the parental KnotBody (without diversification).

Primers described are listed in Table 22. KB_A07 scFv fragment 1 (569 bp) was created by PCR with forward primer 2985 and reverse primer 2999 and DNA template pIONTAS1 harbouring the KB_A07 gene. KB_A07 scFv fragment 2 was created by PCR with primers 2979 and 2980 in the absence of template. Here the primers simply annealed and extended to yield the 137 bp KB_A07 fragment 2. KB_A07 scFv fragment 3 (170 bp) was created by PCR with forward primer 3000 and reverse primer 2995 and DNA template pIONTAS1 harbouring the KB_A07. KB_A12 scFv fragment 1 (572 bp) was created by PCR with forward primer 2985 and reverse primer 3003 and DNA template pIONTAS1 harbouring the KnotBody A12. KB_A12 scFv fragment 2 was created by PCR with primers 2983 and 2984 in the absence of template. Here, the primers simply annealed and extended to yield the 137 bp A12 fragment 2. KB_A12 scFv fragment 3 (179 bp) was created by PCR with forward primer 3004 and reverse primer 3002) and DNA template pIONTAS1 harbouring the KB_A12. Fragments 2 and 3 were gel purified and fragment 1 was purified by spin column. The three fragments were then combined (100 nM each in 10 mM Tris-HCl (pH8)) in a total volume of 10 μl, To this was added 10 μl of KOD Hot Start Master Mix (Merck Millipore, catalogue number 71842). The fragments were then assembled by incubation at 95° C. for 2 minutes followed by 20 cycles of 95° C. (20 s), 60° C. (1 min, 40 s) and 70° C. (30 s). 1 μl of this assembly reaction product was then used as a template in a PCR reaction mix with the outer primers 2985 and 2995 (A07) or 2985 and 3002 (A12). The assembled PCR products encoding the KB_A07 and KB_A12 scFv genes were then digested with NcoI and NotI, gel purified and cloned into the mammalian display targeting vector pD6 (FIG. 26) pre-digested with the same enzymes. Transfection quality plasmid DNA was prepared using the NucleoBond Xtra Midi plus kit (Macherey Nagel, Cat. No. 740412.50).

Electroporation is an efficient way of introducing DNA into cells and the protocols developed by Maxcyte combine high efficiency transfection of mammalian cells combined with high cell viability. HEK293 cells were centrifuged and re-suspended in a final volume of 10⁸ cells/ml in the manufacturer's electroporation buffer (Maxcyte Electroporation buffer, Thermo Fisher Scientific Cat. No. NC0856428)). An aliquot of 4×10⁷ cells (0.4 ml) was added to an OC-400 electroporation cuvette (Maxcyte, Cat. No. OC400R10) with 88 μg DNA (i.e., 2.2 μg/10⁶ cells). The DNA mix consisted of 80 μg of plasmid DNA encoding AAVS-SBI TALENs (pZT-AAVS1 L1 and pZT-AAVS R1 Systems Bioscience Cat. No. GE601A-1) as an equimolar mix and 8 μg of the “donor” plasmid pD6 DNA encoding the KB_A07 or KB_A12. Control electroporations were also performed where the TALEN or donor DNA was replaced by pcDNA3.0. Electroporations were performed according to the manufacturer's instructions (Maxcyte). 2 days after transfection (2dpt) blasticidin selection was initiated (5 μg/ml). After 13 days (15 dpt) cells were analysed by flow cytometry for scFv-Fc expression using an anti-Fc phycoerythrin (PE) labelled antibody. The presence of correctly folded knottin was detected by staining the cells with biotinylated trypsin pre-conjugated with allophycocyanin (APC) labeled streptavidin. Analysis was focused on viable cells using forward scatter and 7AAD staining in the FL3 channel. Cells positive for staining in the FL3 channel (representing non-viable cells which took up 7-AAD) were excluded.

Purified trypsin was biotinylated using EZ-Link Sulfo-NHS-LC-Biotin kit (Pierce Cat. No. 21327) according to manufacturer's instructions. Biotinylated trypsin (1.25 μl, 0.1 mg/ml, 4.3 μM) and streptravidin-APC (Molecular Probes, Cat. No. SA1005, 1 μl, 0.2 mg/ml, 3.8 μM) were pre-incubated in PBS/1% BSA (100 μl) for 30 min at room temperature. Cells (10⁶) per sample, pre-washed with PBS, were re-suspended in the biotinylated trypsin/streptavdin-APC mix prepared above and to this was added anti-human Fc-PE (BioLegend, Cat#409304, 200 μg/ml, 0.5 μl) and incubated for 30 minutes at 4° C. The cells were washed twice in PBS/0.1% BSA, re-suspended in PBS/0.1% BSA containing 7-AAD Viability Staining Solution (eBioscience, Cat #00-6993-50 and analysed using a flow cytometer (Intellicyt iQUE screener).

FIG. 27 shows that after 13 days of blasticidin selection (15 dpt), 34% and 11% of the KB_A07 and KB_A12 transfected HEK293 cells were dual stained for Fc and trypsin. No staining of untransfected HEK293 cells was observed. This demonstrates that it is possible to display membrane tethered KnotBodies on the surface of mammalian cells where the knottin donor is correctly folded.

Example 16. Insertion of Knottins into Antibody VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3, with Variable Linker Sequences, their Display on Phage and Selection of Functional KnotBodies

Examples 1, 2 and 3 demonstrated knottin insertion into antibody VL CDR1 and VL CDR2, display of the resultant KnotBody on the surface of phage and the selection of functional knotbodies. In Examples 1, 2 and 3 libraries were created where variable fusion sequences were introduced between the donor and recipient which were then selected by phage display and screened to identify the optimal fusion sequences for the display of functional knottins. This identified short linker sequences, which were superior to long flexible linkers for the display of functional knottins, as described in Example 11. Insertion of a knottin into VL CDR1 or VL CDR2 enables additional binding contributions from VL and VH CDRs. For some applications there may be an advantage in alternative configurations where the knottin is inserted into alternative CDRs on either VH or VL. It is likely, as described in Example 3, that only a fraction of variable fusion sequences, linking the donor knottin, to each CDR enable the functional display of knottins. To demonstrate that it is possible to display functional knottin in all the antibody CDRs, variants of D1A12 formatted as a scFv (Tape, C. J. et al. (2011) PNAS USA 108, p 5578-5583), were designed with knottin insertions in VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3 where the two codons flanking the knottin were randomised to enable the creation of “linker library” knottin constructs as shown in FIG. 28. The sequence of the parental anti-TACE scFv¹⁰¹ is shown in Table 23. In addition, a linker library was created for KB_A07 where the EETI-II donor knottin was inserted into VL CDR2 as shown in FIG. 28. The inserts encoding the knottins, with randomised linker sequences, inserted into each of the six CDRs of D1A12, and a linker library of KB_A07 where the knottin with randomised flanking amino acids was inserted into VL CDR2, was created by a three fragment assembly as described in Example 15, using standard molecular biology techniques known to those skilled in the art e.g. Schofield et al 2007²⁸. Phage display libraries were created by cloning into the phage display vector pSANG4 (Schofield et al, 2007), phage rescued, as described in Example 3, selected for binding to trypsin and monoclonal phage ELISA was performed with individual clones. Positive clones were obtained demonstrating that functional display of knottin is possible within all the CDR loops in both the antibody variable heavy and light chains.

The strategy and methodology for the preparation of the scFv knottin linker library inserts by a three fragment PCR assembly is detailed in Example 15 and the primers to create the inserts are listed in Tables 22 and 24. The primer pairs required to create the three fragments for each of the libraries, the DNA template (if required) and product sizes are listed in Table 25 and the method to prepare and purify the individual inserts is described in Example 15. The three fragments for each of the libraries A to G, listed in Table 25, were then combined in an equimolar ratio (e.g. fragments A1, A2 and A3 were combined to create library A) and assembled as described in example 15. It was possible to create fragment E (where diversification was near the end of the scFv gene) using a 2 fragment assembly. The products were amplified by PCR with the forward primer 2985 and either reverse primers D1A12 NotI Rev (libraries A to F) or 2995 (library G). The assembled PCR products encoding libraries A to G were then digested with NcoI and NotI, gel purified and ligated into the phage display vector pSANG4 (pre-digested with the same enzymes) and the ligated product electroporated into E. coli TG1 cells (Lucigen, Cat. No. 60502-2) as described in Example 2. Library sizes of between 0.7 to 1.6×10⁸ were achieved. Phage was rescued from the libraries as described in Example 3 to create seven libraries: library A (knottin linker library inserted into D1A12 VH CDR1); library B (knottin linker library inserted into D1A12 VH CDR2); library C (knottin linker library inserted into D1A12 VH CDR3); library D (knottin linker library inserted into D1A12 VL CDR1); library E (knottin linker library inserted into D1A12 VL CDR2); library F (knottin linker library inserted into D1A12 VL CDR3); and library G (knottin linker library inserted into KB_A07 VL CDR2).

To isolate functional knottin-antibody fusions, each of the knottin-antibody linker phage display libraries A to G, described above, were subject to two rounds of phage display selection with biotinylated trypsin as described in Example 3. Individual clones (46 per selection) were picked into 96 well culture plates, phage produced and the phage supernatant from each clone was tested for binding to biotinylated trypsin immobilised on Streptavidin coated Maxisorp™ plates with binding detected using a TRF assay as described in Example 3 (using a coating concentration of biotinylated trypsin of 2.5 μg/ml). The ELISA signals on each 96-well plate were background subtracted with the signal obtained from the average of two negative control readings on the same plate where no phage was added to the well. Specificity for trypsin was confirmed by lack of binding to Streptavidin coated Maxisorp™ in the absence of added trypsin. Screening of clones picked from the unselected knottin linker antibody libraries C, E and G gave 0%, 1% and 32% positive clones respectively indicating that a limited proportion of library members are capable of displaying a functional knottin donor. The hit-rates of clones obtained after phage display selection and monoclonal phage ELISA for libraries A, B, C, D, E, F and G was 11%, 17%, 28%, 4%, 74%, 24% and 93% respectively, demonstrated an enrichment for KnotBodies that display a functional knottin.

Positive clones were sequenced to determine the flanking linker sequences that enabled the display of functional knottins with VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3. The sequences of the linkers sequences identified are listed in Tables 26 and 27. Examples of functional KnotBodies with unique linker sequences were isolated (Table 26) thus demonstrating the insertion of functional knottins into antibody VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3.

Example 17. Alternative Framing of Knottins Inserted into Antibody VL CDR1 and VL CDR2 Domains

In the examples above the native N terminus and C terminus of cys-rich peptides (hereafter referred to as knottins irrespective of structure and disulphide bond pattern) are inserted into an antibody CDR residues in a way that retains the natural linear sequence of the knottin with native N and C termini fused to the antibody frameworks. Alternative “framing” strategies between the recipient antibody and donor knottin are possible however and this is exemplified here. In the following description, the sequences between sequential cysteines in the donor are referred to as “loops” and are numbered 1-5 based on their natural order. For example loop 1 extends between the first cysteine and the second cysteine. Loop 5 extends between the 5^(th) and 6^(th) cysteine. Some cysteine-rich peptides, such as cyclotides are found in a cyclic form where the N and C termini are joined by a peptide sequence. For example MoCoT can be found in a “linear” form (i.e. with free N and C termini) or a cyclic form. The additional peptide sequence joining the first (N terminal) and 6^(th) (C terminal) cysteine in the cyclic form is referred to as “loop 6” (Jagadish et al, (2010) Peptide Science 94 p 611-616) and is used in the examples below to join the original N and C termini.

From one perspective the N and C termini of the donor knottin in a KnotBody are joined by an antibody which might be considered the equivalent of “loop 6” “joining” the N and C terminal cysteines in a cyclic knottin. From that same perspective one could consider alternative “framings” where the antibody replaces alternative loops. In this arrangement the native N and C termini of the donor could be linked by an additional “loop 6” peptide as found in cyclic MoCoTI-II. These alternative framing strategies would allow variation in the juxtaposition of different loops relative to the antibody recipient with potential benefits in creation of optimal bi-specific orientations and in engineering strategies e.g. improving affinity or specificity through contact of both donor and recipient with target proteins or complexes. The effective “rotation” of the donor relative to the recipient could increase availability of certain critical loops eg the trypsin binding loop in EET 5-3 is moved to a more central location within the donor relative to EET 1-5 (see below). The addition of an alternative “loop 6” further increases the possibilities for additional contact with target proteins. Finally such framing alternatives could provide superior expression or folding compared to the native KnotBody construct in some cases.

When a knottin is inserted in its native configuration as described in earlier examples the N terminal segment of the antibody recipient is followed by the first cysteine and loop 1 of the donor knottin. The donor ends at loop 5 followed by the terminal cysteine. In the nomenclature adopted here this is referred to as a “1-5” configuration, representing the order of knottin loops 1, 2, 3, 4, 5. In this case the antibody may be considered as the equivalent of loop 6 of a cyclic knottin. Table 28 illustrates different potential strategies for altering the framing between a knottin and antibody. In Table 28, the natural loop 1 is underlined in each case. The original N and C termini of EETI-II are joined to create “loop 6” and the peptide sequence of loop 6 of MoCOTI-II is used (represented in italics). A more detailed representation of the arrangement of loops in the EET_5-3 configuration is given in FIG. 29A. This shows that in alternative configuration EET_5-3 the order of loops is loop 5, 6, 1, 2 and 3 (with the antibody “replacing” loop 4).

To exemplify selection of knottins inserted in an alternative frame, a knottin donor with framing in a EET_5-3 configuration was fused into either the CDR1 or CDR2 position of either kappa or lambda VL domains. This was done by replacing residues within the VL recipient with random amino acids in a way that limited the number of added amino acids between the 2 domains (FIG. 29B).

Insertion of Knottins with Modified Framing into CDR1

A synthetic EETI-II Knottin donor gene with 5-3 framing was made and was used to replace the CDR1 positions in the light chain recipient by creating a PCR fragment with Pml1 and Mfe sites (which were introduced by PCR) and cloning into pIONTAS1_KB_Kappa and pIONTAS1_KB_lambda. Framework and CDR designations are as described by the International Immunogenetics Information System (IMGT)²⁹. The PCR primers also introduced variable amino acid sequences between the restriction sites and the knottin gene. The PCR primers Pml5-3EETa and Pml5-3EETb each introduced 3 variable codons encoded by the sequence VNS (V=A, C or G, N=A, C, G or T, S=C or G) immediately after the Pml restriction site. The “VNS” degenerate codons encode 16 amino acids (excluding cysteine, tyrosine, tryptophan, phenylalanine and stop codons) at each randomised position from 24 codon combinations. Pml5-3EETb also introduces an additional glycine residue between donor and recipient compared to Pml5-3EETa.

Pml5-3EETa (SEQ ID NO: 79) GTGT VNS VNS VNS TGC GGT CCG AAC GGC TTC TGC Pml5-3EETb (SEQ ID NO: 80) GTGT gga VNS VNS VNS TGC GGT CCG AAC GGC TTC TGC

The 5′ end of these sequences represents the site created by Pml1, a restriction enzyme recognising the sequence “CACGTG” to create a blunt end. To facilitate blunt end cloning of the PCR product primers with 5′ phosphorylated termini were synthesised.

At the 3′ end of the knottin gene the antisense primers 5-3EETMfe (encoding an Mfe1 site) was used to amplify the 5-3 frame of EETI-II. The Mfe1 site used for cloning encodes the 2 and 3rd amino acids of FW2 (Asn-Trp). This primer introduces 2 random amino acids encoded by VNS or VNC codons followed by an Mfe1 site (underlined).

5-3EETMfe (SEQ ID NO: 81) GTCAGTCCAATTGNBSNBGCAGCCGGCCAGGCAGTCTGA

FIG. 29C depicts the sequence following insertion of a 5-3 formatted EETI-II into CDR1 of the lambda light chain IGKV1D-39 using primer Pml5-3EETa. A repertoire of light chain fragments encompassing Framework 2-framework 4 was also cloned after the knottin donor as previously described in example 2 using restriction sites Mfe1 and Not1 (underlined).

Insertion of Knottins into CDR2

The knottin donor was introduced into the CDR2 positions of pIONTAS1_KB_lambda and pIONTAS1_KB_Kappa by creating a PCR fragment with Pst1 and BspE1 sites which were introduced by PCR. It was possible to introduce a Pst1 site into the first two residues of the CDR2 region of the IGKV1 D-39 V kappa gene (within the Ala-Ala sequence). For convenience in cloning the same restriction site and encoded residues were introduced at the end of framework 2 of the V lambda germline gene IGLV1-36. In the lambda germline it was also possible to introduce a BspE1 site into the 4th and 5th residues of the FW3 region encoding Ser-Gly) (FIG. 3E). Again this restriction site and encoded amino acids were also introduced into the V kappa gene (FIG. 3F).

The PCR primers PST5-3EETa was used to amplify EETI-II and introduces 2 Ala codons encompassing a Pst1 site for cloning (underlined) followed by 2 variable codons encoded by the sequence GNS (encoding Val, Ala, Asp or Gly) and VNS (encoding 16 amino acids within 24 codons) immediately after the Pst1 restriction site and preceding the knottin sequence. The VNS codon replaces the first amino acid of EETI-II with the net effect of adding 3 amino acids (2 Ala residues and one of Val, Asp, Ala or Gly) between the donor and recipient framework (FIG. 29 B, D).

PST5-3EETa (SEQ ID NO: 82) ATC TAT GCT GCA GNS VNS TGC GGT CCG AAC GGC TTC TGC

At the 3′ end of the incoming knottin donor, The PCR primers 5-3EETBse was used to amplify the 5-3 framed EETI-II. This introduces a BspE1 site for cloning. The PCR product introduces the knottin donor followed by the glycine which naturally follows the last cysteine of EETI-II (FIG. 3a ). This is followed by 2 randomised amino acids which adjoin to the 4th and 5th amino acids of FW3 after cloning. This has the effect of replacing the first three residues of FW3 with two randomised amino acids and retains the terminal glycine of the knottin (FIG. 3B).

5-3EETBse (SEQ ID NO: 83) GTCAGAGACTCCGGASNBSNBCCCGCAGCCGGCCAGGCAGTCTGA

A repertoire of light chain fragments encompassing Framework 3-framework 4 was also cloned after the knottin donor as previously described in example 2.

FIG. 29D depicts the sequence following insertion of a 5-3 formatted EETI-II into CDR2 of the lambda light chain IGKV1D-39 using primer 5-3EETBse. A repertoire of light chain fragments encompassing Framework 3-framework 4 was also cloned after the knottin donor as previously described in example 2 using restriction sites BspE1 and Not1.

Using standard molecular biology techniques known to those skilled in the art e.g. Schofield et al 2007²⁸, constructs (as depicted in FIGS. 29B, C and D)) were created using gene fragments created as described above. These were cloned into pIONTAS1_KB_Kappa” and “pIONTAS1_KB_lambda and were electroporated into E. coli TG1 cells and KnotBody library sizes of 3.5-6.6×10⁸ members were constructed

Following selection on trypsin, as described in example 3, positive clones were identified by monoclonal phage ELISA and their sequences determined. Examples of positive clones in CDR1 or CDR 2 of kappa and lambda light chains are shown in Table 29 with the linker sequences shown in lower case and the knottin donor in a 5-3 frame shown in italics. Also shown is the value of ELISA signal attained from monoclonal phage ELISA. The specificity of interaction was further confirmed by mutating the pair of Proline-Arginine residues involved in interaction with trypsin to alanine-alanine which diminished binding (as described in example 3).

Example 18: Evaluating the Immunomodulatory Function of Anti-Kv1.3 KnotBodies

Kv1.3 plays a key role in maintenance of calcium signalling following activation of T cell receptors thereby modulating T cell activation, proliferation and cytokine release in effector memory T (T_(EM)) cells. T_(EM) cells play an important role in the pathogenesis of T lymphocyte mediated autoimmune diseases and so blockade of Kv1.3 activity represents a potential point of therapeutic intervention.

Example 7 describes the generation of Kv1.3 blockers by replacing the trypsin binding knottin EETI-II at the CDR2 position of KnotBodies KB_A07 and KB_A12 with the cysteine rich miniproteins Shk or Kaliotoxin (naturally occurring Kv1.3 blockers from sea anemone venom and scorpion venom respectively). Example 8 demonstrates the blocking activity of these KnotBodies on Kv1.3 determined by electrophysiology. In this example, we assess the ability of KB_A12 Shk to modulate the cytokine secretion by activated T cells.

Methods for measuring cytokine release from T cells are well known to those skilled in the art (eg Tarcha et al, (2012) J Pharmacology and Experimental Therapeutics, 342 p 642-653). Peripheral blood mononuclear cells (PBMCs) were isolated from 5 healthy donors and were simulated by culturing in 96-well plates pre-coated with 500 ng/ml of the anti-CD3 antibody OKT3 (ebioscience, Cat. No. 16-0037-85). PBMCs were suspended at 2×10⁶ cells/ml and 100 ul of cells suspension mixed with 100 ul of test sample (KB_A12_ShK) or positive control (free Shk toxin, Alomone labs, STS-400) or negative control (parental KnotBody KB_A12) at a final concentration of 100 nM. (Samples were in triplicate for KB_A12_Shk or duplicate for KB_A12 and free Shk). Cells were incubated at 37° C. for 72 hours, centrifuged at 1500 rpm for 5 mins at room temperature before removing 150 ul of supernatant. After 72 hours incubation, the concentration of cytokines and Granzyme-B was determined using a Luminex bead based assay according to manufacturers instructions (Thermo Fisher).

Secretion of Interferon gamma, Granzyme B, interleukin-17A and TNF-alpha was reduced in PBMCs from all 5 different donors when incubated with KB_A12_Shk compared to controls incubated with a control KnotBody (KB_A12) (FIGS. 30A to D). This demonstrates that KB_A12_ShK not only blocks Kv1.3 function as measured by electrophysiology (example 8) but also directly interferes with T cell activation as determined in a T cell functional assay.

TABLE 1 Sequence of unique EETI-II CDR1 and CDR2 KnotBody binders derived from EETI-II CDR1 and CDR2 libraries Clone ID Sequence CDR1_F_10 QSVLTQPPSVSEAPRQRVTITCGERPCPRILMRCKQDSDCLAGCVCGPNGFCGANNWYQ QLPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNQ VFGGGTKVTVLGQP (SEQ ID NO: 84) CDR1_B_01 QSVLTQPPSVSEAPRQRVTITCGGGRCPRILMRCKQDSDCLAGCVCGPNGFCGTPNWYQ QLPGSSPTTLIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTGDEADYYCQSYDSSNQ VFGGGTQLTVLGQP (SEQ ID NO: 85) CDR1_F_02 QSVLTQPPSVSEAPRQRVTITCGSRPCPRILMRCKQDSDCLAGCVCGPNGFCGSHNWYQ QLPGSSPTTVIYEDNQRPSGVPDRFSGSIDRSSNSASLTISGLKTEDEADYYCQSYDSSNH RVVFGGGTKLTVLGQP (SEQ ID NO: 86) CDR1_F_05 QSVLTQPPSVSEAPRQRVTITCGGGRCPRILMRCKQDSDCLAGCVCGPNGFCGTGNWYQ QLPGRSPTNVVYEDNQRPPGVSDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNQ VFGGGTKLTVLGQP (SEQ ID NO: 87) CDR1_C_12 QSVLTQLPSVSEAPRQRVTITCGRAMCPRILMRCKQDSDCLAGCVCGPNGFCGTGNWYQ QHPGKAPKLIIFDVSKRPSGVPDRFSASKSGNTASLTISGLQAEDEADYYCNSYTSSNTWVF GGGTQLTVLGQP (SEQ ID NO: 88) CDR1_G_05 QSVLTQPPSVSEAPRQRVTITCDRKCPRILMRCKQDSDCLAGCVCGPNGFCGTTNWYQQL PGSSPTTVIYENFQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSYNQVFG SGTKLTVLGQP (SEQ ID NO: 89) CDR1_C_06 QSVLTQPPSVSEAPRQRVTITCGRRGCPRILMRCKQDSDCLAGCVCGPNGFCGGDNWYQ QHPGSSPTPVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKPEDEADYYCQSHDGSNP WVFGGGTKLTVLGQP (SEQ ID NO: 90) CDR1_A_03 QSVLTQPPSVSEAPRQRVTITCGGRCPRILMRCKQDSDCLAGCVCGPNGFCGSANWYQQ LPDSAPATVIYEDNQRPSGVPDRFSGSIDTSSNSASLTISGLKTEDEADYYCQSYDSSNHW VFGGGTKLTVLGQP (SEQ ID NO: 91) CDR1_D_06 QSVLTQPPSVSEAPRQRVTITCRGGCPRILMRCKQDSDCLAGCVCGPNGFCGSPNWYQQ KPGQAPVLVIYGKNNRPSGIPDRFSGSRSGTPASLTITGAQAEDEADYYCNSRDNDNNHVV FGGGTKLTVLGQP (SEQ ID NO: 92) CDR1_E_08 QSVLTQPPSVSEAPRQRVTITCGGTGCPRILMRCKQDSDCLAGCVCGPNGFCGSANWYQ QLPGSSPTNVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSNDGTNG VFGGGTKVTVLGQP (SEQ ID NO: 93) CDR1_A_12 QSVLTQPPSVPEAPRQRVTITCGARPCPRILMRCKQDSDCLAGCVCGPNGFCGASNWYQ QLPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSINRG VFGGTQLTVLGQP (SEQ ID NO: 94) CDR1_H_03 QSVLTQPPSVSEAPRQRVTITCGRTGCPRILMRCKQDSDCLAGCVCGPNGFCGTVNWYQ QLPGSAPTTVIYEDNQRPSGVPDRFSGPIDSSSNSAPLTISGLKTEDEADYYCQSYDRNNVI FGGGTKLTVLGQP (SEQ ID NO: 95) CDR1_E_10 QSVLTQPPSVSEAPRQRVTITCGTRGCPRILMRCKQDSDCLAGCVCGPNGFCGSNNWYQ QLPGSSPTTVIYEDNQRPSGVPDRFYGSIDSSSDSASLTISGLETEDEADYFCHSYDSDKW VFGGGTQLTVLGQP (SEQ ID NO: 96) CDR1_E_03 QSVLTQPPSVSEAPRQRVTITCGGMPCPRILMRCKQDSDCLAGCVCGPNGFCGATNWYQ QLPGSSPTTVIYEDNQRPSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSYDSSNPK VVFGGGTKLTVLGQP (SEQ ID NO: 97) CDR2_D_02 QSVLTQPPSVSEAPGQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAAKCPRILMRC KQDSDCLAGCVCGPNGFCGTRSGVSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDD SLSGYVFGTGTKLTVLGQP (SEQ ID NO: 98) CDR2_D_09 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAADKCPRILMRC KQDSDCLAGCVCGPNGFCGGGSGIPERFSGSKSGTSASLAISGLRSEDEADYYCATWDDN LNGVVFGGGTKLTVLGQP (SEQ ID NO: 99) CDR2_H_09 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAVGCPRILMR CKQDSDCLAGCVCGPNGFCGTGSGIPERFSGSKSGNTASLTISGLQAEDEGDYYCAAWDD SLSGPVFGGGTKLTVLGQP (SEQ ID NO: 100) CDR2_F_02 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAGRCPRILMR CKQDSDCLAGCVCGPNGFCGARSGIPERFSASKSGTSASLVISGLQSEDEADYYCAAWDD SLNGWVFGGGTKLTVLGQP (SEQ ID NO: 101) CDR2_C_07 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAAVCPRILMRC KQDSDCLAGCVCGPNGFCGTRSGIPERFSGSKSGTSAFLAISGLRSEDEADYYCAAWDDS LSGVVFGGGTKLTVLGQP (SEQ ID NO: 102) CDR2_B_07 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAARCPRILMRC KQDSDCLAGCVCGPNGFCGHTSGIPERFSGSKSGTSASLAISGLQSEDEADYYCAAWDDS LNGVVFGGGTKLTVLGQP (SEQ ID NO: 103) CDR2_G_05 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAGRCPRILMR CKQDSDCLAGCVCGPNGFCGGRSGVSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD DSLNGYVFGTGTKLTVLGQP (SEQ ID NO: 104) CDR2_H_04 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAGRCPRILMR CKQDSDCLAGCVCGPNGFCGANSGVSDRFSAAKSGTSASLAINGLRSEDEADYYCAAWD DSLNGYVFGTGTKLTVLGQP (SEQ ID NO: 105) CDR2_A_01 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAEPCPRILMRC KQDSDCLAGCVCGPNGFCGAPSGVPDRFSGSKSGTSASLAITGLQSEDEAHYYCAAWDD SLSAWVFGGGTKLTVLGQP (SEQ ID NO: 106) CDR2_H_07 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAAGCPRILMR CKQDSDCLAGCVCGPNGFCGTRSGVSDRFSGSKSGTSASLAISGLRSEDEADYYCAAWD DSLRAYVFGTGTKLTVLGQP (SEQ ID NO: 107) CDR2_B_06 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAADRCPRILMR CKQDSDCLAGCVCGPNGFCGTDSGIPERFSGSKSGNTASLTISGLQAEDEADYYCAAWDD SLSGPVFGGGTKVTVLGQP (SEQ ID NO: 108) CDR2_F_09 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAVICPRILMRC KQDSDCLAGCVCGPNGFCGTGSGVSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDD SLNGVVFGGGTKLTVLGQP (SEQ ID NO: 109) CDR2_F_11 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAERCPRILMRC KQDSDCLAGCVCGPNGFCGGSSGVSDRFSGSKSGTSASLAISGLRSEDEADYYCATWDD NLNGPVFGGGTKLTVLGQP (SEQ ID NO: 110) CDR2_C_02 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAVGCPRILMR CKQDSDCLAGCVCGPNGFCGSASGVSDRFSGSKSGTSASLAISGLRSEDEADYYCASWD DSLRGYVFGTGTKVTVLGQP (SEQ ID NO: 111) CDR2_D_08 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAGPCPRILMR CKQDSDCLAGCVCGPNGFCGTASGVPDRFSGSKSGTSASLAISGLRSEDEADYYCAAWD DSLSGVVFGGGTKVTVLGQP (SEQ ID NO: 112) CDR2_E_10 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAARCPRILMRC KQDSDCLAGCVCGPNGFCGTPSGVPDRFSGSKSGTSASLAISGLQSEDEADYYCAAWDD SLSGYVFGTGTKLTVLGQP (SEQ ID NO: 113) CDR2_F_05 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGSNAVNWYQQLPGKAPKLLIYAAGGCPRILMR CKQDSDCLAGCVCGPNGFCGSNSGVPDRFSGSKSGNTASLTISGLQSEDEADYYCAAWD DSLSGVVFGGGTQLTVLGQP (SEQ ID NO: 114) CDR2_G_04 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAGMCPRILMR CKQDSDCLAGCVCGPNGFCGGHSGVSDRFSGSKSGTSASLAISGLQSEDEADYYCAAWD DSLNGYVFGTGTQLTVLGQP (SEQ ID NO: 115) CDR2_A_08 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAARCPRILMRC KQDSDCLAGCVCGPNGFCGTRSGVSDRFSGSKSGTSASLAISGLQSGDEADYYCAAWDD SLNGWVFGGGTKLTVLGQP (SEQ ID NO: 116) CDR2_E_06 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAADKCPRILMRC KQDSDCLAGCVCGPNGFCGLTSGVSDRFSGSKSGTSASLAISGLRSEDEADYYCATWDDS LNGVVFGGGTKLTVLGQP (SEQ ID NO: 117) CDR2_B_11 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAAKCPRILMRC KQDSDCLAGCVCGPNGFCGGASGVSDRFSGSIDSSSNSASLTISGLKPEDEGDYYCQSYD SSNRWVFGGGTKVTVLGQP (SEQ ID NO: 118) CDR2_G_09 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAARCPRILMRC KQDSDCLAGCVCGPNGFCGTTSGVPDRFSGSIDSSSNSASLTISELKTEDEADYYCQSYDS SNQGWVFGGGTKLTVLGQP (SEQ ID NO: 119) CDR2_C_08 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAGRCPRILMR CKQDSDCLAGCVCGPNGFCGGGSGVPDRFSGSIDSSSNSASLTISGLRAEDEADYYCQSY DSSNHWVFGGGTKLTVLGQP (SEQ ID NO: 120) CDR2_E_09 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAGPCPRILMR CKQDSDCLAGCVCGPNGFCGPRSGVPDRFSGSIDSSSNSASLTISGLKTEDEADYYCQSY DSNNHWVFGGGTKLTVLGQP (SEQ ID NO: 121) CDR2_F_04 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAERCPRILMRC KQDSDCLAGCVCGPNGFCGTPSGVPDRFSGSIDTFSNSASLTISGLKTEDEADYYCQSYDS SHHWVFGGGTKLTVLGQP (SEQ ID NO: 122) CDR2_E_05 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAGPCPRILMR CKQDSDCLAGCVCGPNGFCGADSGVSDRFSGSKSGTSASLAITGLQAEDEGDYYCAAWD DSLNGLVFGGGTKVTVLGQP (SEQ ID NO: 123) CDR2_D_07 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAGRCPRILMR CRQDSDCLAGCVCGPNGFCGTASGVPDRFSGSIDTFSNSASLTISGLKTEDEADYYCQSY DSSNHWVFGGGTKLTVLGQP (SEQ ID NO: 124) CDR2_G_03 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAGRCPRILMR CKQDSDCLAGCVCGPNGFCGTNSGIPERFSGSKSGNTASLTISGLQAEDEADYYCQSYDS SLSGWVFGGGTQLTVLGQP (SEQ ID NO: 125) CDR2_A_09 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAAPCPRILMRC KQDSDCLAGCVCGPNGFCGAHSGVPDRFSGSIDRSSNSASLTISGLKIEDEADYYCQSYDS SNHVVFGGGTKVTVLGQP (SEQ ID NO: 126) CDR2_D_11 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIYAAVRCPRILMRC KQDSDCLAGCVCGPNGFCGTPSGIPERFSGSIDRSTNSASLTISGLKTEDEADYYCQSYDS SNLNWVFGGGTKLTVLGQP (SEQ ID NO: 127) CDR2_C_06 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLINAADRCPRILMR CKQDSDCLAGCVCGPNGFCGTNSGVPDRFSGSSSGNTASLTITGAQAEDEADYYCNSRD SSGNNWVFGGGTKVTVLGQP (SEQ ID NO: 128) CDR2_F_01 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAAGLCPRILMRCK QDSDCLAGCVCGPNGFCGGESGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP YTFGQGTKVDIKRT (SEQ ID NO: 129) CDR2_F_12 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAAAPCPRILMRCK QDSDCLAGCVCGPNGFCGSTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP YTFGQGTKVDIKRT (SEQ ID NO: 130) CDR2_E_04 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAAARCPRILMRCK QDSDCLAGCVCGPNGFCGTTSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP YTFGQGTKVEIKRT (SEQ ID NO: 131) CDR2_F_03 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAAAKCPRILMRCK QDSDCLAGCVCGPNGFCGAPSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP YTFGQGTKVEIKRT (SEQ ID NO: 132) CDR2_D_03 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAAVVCPRILMRCK QDSDCLAGCVCGPNGFCGSGSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP YTFGQGTKLEIKRT (SEQ ID NO: 133) CDR2_D_06 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAAGRCPRILMRCK QDSDCLAGCVCGPNGFCGTASGVPSRFSGGGSGTDFTLTISSLQPEDFATYYCQQSYSTP YTFGQGTKVDIKRT (SEQ ID NO: 134) CDR2_C_09 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAAGGCPRILMRCK QDSDCLAGCVCGPNGFCGAPSGVPSRFSGSGSGTDFTLTISGLQPEDFGTYYCQQSYSTP LTFGGGTKLEIKRT (SEQ ID NO: 135) CDR2_E_08 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAAGVCPRILMRCK QDSDCLAGCVCGPNGFCGSRSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGASPP YTFGQGTKVEIKRT (SEQ ID NO: 136) CDR2_G_11 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAAGRCPRILMRCK QDSDCLAGCVCGPNGFCGGHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSYSTP PTFGQGTKLEIKRT (SEQ ID NO: 137) CDR2_B_03 DIQMTQSPSSLSASVGDRVTITCRASQSISGYLNWYQQKPGKAPKLLIYAAEVCPRILMRCK QDSDCLAGCVCGPNGFCGGTSGVPSRFSGSRSGTEFTLTISSLQPEDFATYYCQKVDDYP LTFGGGTKVEIKRT (SEQ ID NO: 138) CDR2_H_02 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAAEICPRILMRCKQ DSDCLAGCVCGPNGFCGPSSGIPARFSGSGYGTDFTLTINNIESEDAAYYFCLQHDNFPYT FGQGTKVEIKRT (SEQ ID NO: 139)

TABLE 2 KnotBodies selected for detailed characterisation. C- Tryspin terminal Clone binding N-terminal linker linker number Clone ID Library signal (FU) sequence sequence 1 KB_A01 CDR2_B_03 20173 EV GT 2 KB_A02 CDR2_B_11 183150 AK GA 3 KB_A03 CDR2_C_06 28711 DR TN 4 KB_A04 CDR2_D_03 47015 VV SG 5 KB_A05 CDR2_D_09 121458 DK GG 6 KB_A06 CDR2_D_11 24427 VR TP 7 KB_A07 CDR2_E_08 21448 GV SR 8 KB_A08 CDR2_F_01 167526 GL GE 9 KB_A09 CDR2_F_04 57732 ER TP 10 KB_A10 CDR2_F_05 38794 GG SN 11 KB_A11 CDR2_H_02 16399 EI PS 12 KB_A12 CDR2_H_04 69349 GR AN 13 KB_B01 CDR2_H_07 57667 AG TR 14 KB_B02 CDR2_H_09 115848 VG GTG 15 KB_C07 CDR2_C_02 43304 VG SA 16 KB_C08 CDR2_C_07 76810 AV TR 17 KB_C09 CDR2_C_08 89424 GR GG 18 KB_C10 CDR2_E_05 118631 GP AD 19 KB_B10 CDR1_C_12 9268 GRAM (SEQ ID NO: TG 140) 20 KB_B11 CDR1_F_02 10286 GSRP (SEQ ID NO: SH 141) 21 KB_B12 CDR1_F_10 17356 GERP (SEQ ID NO: AN 142)

TABLE 3 Analysis of trypsin binding of KnotBody GlyAla mutants. Clone % reduction in number Library Clone ID binding signal 1 EETI-II CDR2 KB_A01 93 2 KB_A02 96 3 KB_A03 90 4 KB_A04 100 5 KB_A05 100 6 KB_A06 100 7 KB_A07 98 8 KB_A08 99 9 KB_A09 100 10 KB_A10 100 11 KB_A11 100 12 KB_A12 99 13 KB_B01 95 14 KB_B02 98 15 KB_C07 100 16 KB_C08 100 17 KB_C09 100 18 KB_C10 100 19 EETI-II CDR1 KB_B10 100 20 KB_B11 100 21 KB_B12 97

TABLE 4 Trypsin binding of KnotBody Fabs. TRF signal for TRF signal for TRF signal for Sample ID 10 ug/mL Fab 2.4 ug/mL Fab 0.6 ug/mL Fab Background 420 406 431 (No Fab control) KB_A01 55581 16598 2392 KB_A02 255452 220577 93759 KB_A04 264243 220697 111314 KB_A05 217918 65386 46706 KB_A06 241777 211701 108395 KB_A07 226082 201003 107814 KB_A08 152699 106949 36568 KB_A09 161164 84131 20987 KB_A10 68660 71860 14572 KB_A11 212086 140605 48852 KB_A12 252832 221989 102132 KB_B01 256957 152908 54794 KB_B02 56567 15884 2344 KB_C07 276261 247038 153317 KB_C08 182923 131246 51143 KB_C09 288988 204878 46788 KB_C10 238066 146488 41709

TABLE 5 Bi-specific KnotBodies identified from phage display selections. Selection Number of bi-specific Percentage of bi-specific antigen binders binders cMET-FC 33 68.8 GAS6-CD4 36 75.0 FGFR4-CD4 42 87.5 UPA 48 100.0 B-gal 19 39.6

TABLE 6 KnotBody heavy chain shuffled clones with improved binding to trypsin (HCDR3- SEQ NOs: 143-165) Fold imrpovemnt Fold VH germline Capture in capture assay Improvement Clone ID Parent Clone (DP numbering) Heavy chain CDR3 sequence assay signal signal (x) kd (1/s) in kd (x) KB_Tr_F03 KB_A12 Vh1_DP-8,75_(1-02) HAANWEEPGFDP 359729 8.3 0.0006 4.4 KB_Tr_E05 KB_A12 Vh1_DP-8,75_(1-02) RSSAGDGAYFDY 437719 10.1 0.0011 2.6 KB_Tr_E02 KB_A07 Vh1_DP-10_(1-69) DRGLGTSTYYYGMDV 140056 9.0 0.0013 2.3 KB_Tr_D10 KB_A12 Vh1_DP-15_(1-08) SQSGVGGTGFDP 401740 9.3 0.0014 1.9 KB_Tr_A08 KB_A12 Vh6_DP-74_(6-1) QLGGTFDV 182731 4.2 0.0015 1.8 KB_Tr_E10 KB_A12 Vh1_DP-10_(1-69) ALDGAWTDYGDSHEAYGMDV 125996 2.9 0.0016 1.7 KB_Tr_B08 KB_A12 Vh2_DP-27,28_(2-70) SFDYGDPFDY 209098 4.8 0.0016 1.7 KB_Tr_C12 KB_A12 Vh1_DP-10_(1-69) SGAVGARDAFDI 116225 2.7 0.0016 1.7 KB_Tr_F01 KB_A12 Vh1_DP-8,75_(1-02) DRIGEGIPMDV 278828 6.5 0.0016 1.7 KB_Tr_A10 KB_A12 Vh1_DP-5_(1-24) GGIVGATNDAFDI 74429 1.7 0.0016 1.7 KB_Tr_B02 KB_A12 Vh1_DP-15_(1-08) AGAGEGGMDV 339089 7.9 0.0016 1.7 KB_Tr_H03 KB_A07 Vh1_DP-5_(1-24) DNPLRGMDV 217117 14.0 0.0017 1.7 KB_Tr_A07 KB_A12 Vh3_DP-47_(3-23) ESGGTNGWDAFDM 99305 2.3 0.0017 1.6 KB_Tr_B07 KB_A12 Vh1_DP-10_(1-69) GERPDYYYYYGMDV 218284 5.1 0.0017 1.6 KB_Tr_C04 KB_A12 Vh6_DP-74_(6-1) EPGGDSYFDY 203206 4.7 0.0017 1.5 KB_Tr_D05 KB_A12 Vh1_DP-5_(1-24) MRGYNGGFDL 220428 5.1 0.0018 1.5 KB_Tr_D01 KB_A12 Vh1_DP-88_(1-e) SGGEGVRAYGMDV 227897 5.3 0.0018 1.5 KB_Tr_B03 KB_A12 Vh1_DP-10_(1-69) DGNVRGMDV 168630 3.9 0.0018 1.5 KB_Tr_D03 KB_A12 Vh1_DP-10_(1-69) GGSDYYMDV 237382 5.5 0.0018 1.5 KB_Tr_H07 KB_A12 Vh3_DP-47_(3-23) GDSSGYGSFDI 209783 4.9 0.0018 1.5 KB_Tr_G09 KB_A12 Vh1_DP-10_(1-69) SGGLGDAFDI 237406 5.5 0.0018 1.5 KB_Tr_D07 KB_A07 Vh4_DP-63_(4-34) VSTYSGSQPFDY 205899 13.2 0.0018 1.6 KB_Tr_D09 KB_A12 Vh6_DP-74_(6-1) GAGEGYFDY 320628 7.4 0.0018 1.5

TABLE 7 Ion channel blocking knottins used for antibody VL CDR2 insertion. Target Ion Toxin channel Reference Database Accession Huwentoxin-IV Nav 1.7 71 AAP33074.1 GI:30575584 ProTx-II Nav 1.7 72 P83476.1 GI:25091451 Ssm6a Nav 1.7 70 2MUN_A GI:848130036 Kaliotoxin Kv 1.3 73, 107 AAB20997.1 GI:242975 (KTX) MoKa-1 Kv 1.3 25 2KIR_A GI:282403522 Shk Kv 1.3 74, 109 4LFS_A GI:530537696 Psalmotoxin ASIC1a 118  P60514.1 GI:44888346 (PcTX1) EETI-II n/a 24 P12071.2 GI:547744 Conotoxin-ω Cav 2.2 58-60 1TTK_A GI:253723132 MCoTI-II n/a 119  P82409.1 GI:8928147 Mambalgin-1 ASIC1a/2a/2b 121  P0DKR6 (AFT65615.1) (Mba-1) Mambalgin-2 ASIC1a/2a/2b 121  P0DKS3 (Mba-2)

TABLE 8A DNA and amino acid sequences of ion channel blocking knottins. Toxin Sequence Huwentoxin-IV GAGTGCCTGGAAATCTTCAAGGCCTGCAACCCCAGCAACGACCAGTGCTGC AAGAGCAGCAAGCTCGTGTGCAGCAGAAAGACCCGGTGGTGCAAGTACCAG ATCTCCAGATGACCCAGAGCCCAAGCAGCCTGAGCGCCA ((SEQ ID NO: 7) ECLEIFKA CNPSNDQCCK SSKLVCSRKT RWCKYQIGK (SEQ ID NO: 8) ProTx-II TACTGCCAGAAATGGATGTGGACCTGCGACAGCGAGCGGAAGTGCTGCGAG GGCATGGTGTGCCGGCTGTGGTGCAAGAAAAAGCTGTGGTCCAGATGACCC AGAGCCCAAGCAGCCTGAGCGCCA (SEQ ID NO: 9) YCQKWMWTCD SERKCCEGMV CRLWCKKKLW (SEQ ID NO: 10) Ssm6a GCCGACAACAAGTGCGAGAACAGCCTGCGGAGAGAGATCGCCTGCGGCCA GTGCCGGGACAAAGTGAAAACCGACGGCTACTTCTACGAGTGCTGCACCAG CGACAGCACCTTCAAGAAGTGCCAGGACCTGCTGCACTCCAGATGACCCAG AGCCCAAGCAGCCTGAGCGCCA (SEQ ID NO: 11) ADNKCENSLR REIACGQCRD KVKTDGYFYE CCTSDSTFKK CQDLLH (SEQ ID NO: 12) Kaliotoxin (KTX) ATCTATGCTGCAGGGAGGGGTGTGGAAATTAACGTGAAGTGTAGCGGGAGC CCACAGTGCCTTAAACCATGCAAAGATGCGGGGATGCGCTTTGGAAAGTGCA TGAACCGTAAATGCCACTGCACGCCGAAGGCCAACTCCGGAGTCTC (SEQ ID NO: 13) GVEINVKCSG SPQCLKPCKD AGMRFGKCMN RKCHCTP (SEQ ID NO: 14) Mokatoxin-1 ATCAACGTGAAGTGCAGCCTGCCCCAGCAGTGCATCAAGCCCTGCAAGGAC (MoKa-1) GCCGGCATGAGATTCGGCAAGTGCATGAACAAGAAATGCCGGTGCTACAGC TCCAGATGACCCAGAGCCCAAGCAGCCTGAGCGCCA (SEQ ID NO: 15) INVKCSLPQQ CIKPCKDAGM RFGKCMNKKC RCYS (SEQ ID NO: 16) Shk AGAAGCTGCATCGACACCATCCCCAAGAGCAGATGCACCGCCTTCCAGTGC AAGCACAGCATGAAGTACCGGCTGAGCTTCTGTAGAAAGACCTGCGGCACC TGTTCCAGATGACCCAGAGCCCAAGCAGCCTGAGCGCCA (SEQ ID NO: 17) RSCIDTIPKS RCTAFQCKHS MKYRLSFCRK TCGTC (SEQ ID NO: 18) PcTX1 GAGGACTGCATCCCCAAGTGGAAGGGCTGCGTGAACAGACACGGCGACTGT TGCGAGGGCCTGGAATGCTGGAAGCGGAGGCGGAGCTTCGAAGTGTGCGT GCCCAAGACCCCTAAGACCTCCAGATGACCCAGAGCCCAAGCAGCCTGAGC GCCA (SEQ ID NO: 19) EDCIPKWKGC VNRHGDCCEG LECWKRRRSF EVCVPKTPKT (SEQ ID NO: 20) EETI-II GCPRILMRCK QDSDCLAGCV CGPNGFCGSP (SEQ ID NO: 21) Conotoxin-omega CKGKGAKCSR LMYDCCTGSC RSGKCX (SEQ ID NO: 22) MCoTI-II SGSDGGVCPK ILKKCRRDSD CPGACICRGN GYCG (SEQ ID NO: 23) Mambalgin-1 LKCYQHGKVVTCHRDMKFCYHNTGMPFRNLKLILQGCSSSCSETENNKCCSTD (Mba-1) RCNK (SEQ ID NO: 24) Mambalgin-2 LKCFQHGKVVTCHRDMKFCYHNTGMPFRNLKLILQGCSSSCSETENNKCCSTD (Mba-1) RCNK (SEQ ID NO: 25)

TABLE 8B Sequences of ion channel blocking recipient: donor fusions (knottin sequence underlined) KnotBody domain Sequence D1 A12 (VH) EVQLVESGGGLVRPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEW VSAISGSGGSTYYADSVKGRFTISRDNTKNSLYLQMTSLRADDTAFYYC VKDFGPGYGTGWFDYWGPGTLVTVSS (SEQ ID NO: 30) KB_A07 ShK DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA (VL) AGVRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTCSRSGVPSRFS GSGSGTDFTLTISSLQPEDFATYYCQQGASPPYTFGQGTKVEIKR (SEQ ID NO: 31) KB_A12 ShK QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLI (VL) YAAGRRSCIDTIPKSRCTAFQCKHSMKYRLSFCRKTCGTCANSGVSDR FSAAKSGTSASLAINGLRSEDEADYYCAAWDDSLNGYVFGTGTKLTVLG (SEQ ID NO: 32) KB_A07 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA Kaliotoxin (VL) AGVGVEINVKCSGSPQCLKPCKDAGMRFGKCMNRKCHCTPKSRSGVP SRFSGSGSGTDFTLTISSLQPEDFATYYCQQGASPPYTFGQGTKVEIKR (SEQ ID NO: 33) KB_A12 PcTx1 QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLI (VL) YAAGREDCIPKWKGCVNRHGDCCEGLECWKRRRSFEVCVPKTPKTAN SGVSDRFSAAKSGTSASLAINGLRSEDEADYYCAAWDDSLNGYVFGTG TKLTVLG (SEQ ID NO: 34) KB_A07 PcTx1 DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYA (VL) AGVEDCIPKWKGCVNRHGDCCEGLECWKRRRSFEVCVPKTPKTSRSG VPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQGASPPYTFGQGTKVEI KR (SEQ ID NO: 35)

TABLE 9A Primers used for the cloning of ion channel blockers (toxins) into the VL CDR2 of KB_A07. Primer name identifies the toxin used for the fusion (e.g. Huwen_A07 Rev is a reverse primer used for cloning Huwentoxin-IV into KB_A07) Primer Sequence Huwen_A07 Fwd TATGCTGCAGGGGTCGAGTGCCTGGAAATCTTCAAGGCCTGC (SEQ ID NO: 166) Huwen_A07 Rev TGGGACTCCGGAGCGGCTGATCTGGTACTTGCACCACCGGGT(SEQ ID NO: 167) ProTx-II A07 Rev TGGGACTCCGGAGCGGCTCCACAGCTTTTTCTTGCACCACAG(SEQ ID NO: 168) ProTx_IIA07 fwd TATGCTGCAGGGGTCTACTGCCAGAAATGGATGTGGACCTG(SEQ ID NO: 169) Ssm6_A07 Fwd TATGCTGCAGGGGTCGCCGACAACAAGTGCGAGAACAGCCTG(SEQ ID NO: 170) SSm6_A07 Rev TGGGACTCCGGAGCGGCTGTGCAGCAGGTCCTGGCACTTCTT(SEQ ID NO: 171) Ssm6_GGS_A07 TATGCTGCAGGGGTCGGCGGTAGCGCCGACAACAAGTGCGAGAACAGC Fwd CTG(SEQ ID NO: 172) Ssm6_GGS_A07 GACTCCGGAGCGGCTACCGCCGCTGTGCAGCAGGTCCTGGCACTTCTT Rev GAA(SEQ ID NO: 173) Kalio_A07_Fwd_B ATCTATGCTGCAGGGGTCGGTGTGGAAATTAACGTGAAGTGT(SEQ ID NO: 174) Kalio_A07 Rev TGGGACTCCGGAGCGGCTCTTCGGCGTGCAGTGGCATTTACG(SEQ ID NO: 175) Moka-1 A07 Fwd TATGCTGCAGGGGTCATCAACGTGAAGTGCAGCCTGCCCCAG(SEQ ID NO: 176) Moka-1 A07 Rev TGGGACTCCGGAGCGGCTGCTGTAGCACCGGCATTTCTTGTT(SEQ ID NO: 177) Shk_A07 Fwd TATGCTGCAGGGGTCAGAAGCTGCATCGACACCATCCCCAAGA(SEQ ID NO: 178) Shk_A07 Rev TGGGACTCCGGAGCGGCTACAGGTGCCGCAGGTCTTTCTACA(SEQ ID NO: 179) PcTx-I A07 Fwd TATGCTGCAGGGGTCGAGGACTGCATCCCCAAGTGGAAGGGC(SEQ ID NO: 180) PcTx-I A07 Rev TGGGACTCCGGAGCGGCTGGTCTTAGGGGTCTTGGGCACGCA(SEQ ID NO: 181)

TABLE 9B Primers used for the cloning of ion channel blockers (toxins) into the VL CDR2 of KB_A12. Primer name identifies the toxin used for the fusion (e.g Huwen_A12 Fwd is a forward primer used for cloning Huwentoxin-IV into KB_A07). Primer Sequence Huwen A12 Rev GACTCCGGAGTTGGCGATCTGGTACTTGCACCACCGGGTCTTTCT(SEQ ID NO: 182) Huwen_A12 Fwd TATGCTGCAGGGAGGGAGTGCCTGGAAATCTTCAAGGCCTGC(SEQ ID NO: 183) ProTx-II A12 fwd TATGCTGCAGGGAGGTACTGCCAGAAATGGATGTGGACCTGC(SEQ ID NO: 184) ProTx-II A12 Rev GACTCCGGAGTTGGCCCACAGCTTTTTCTTGCACCACAGCCG(SEQ ID NO: 185) Ssm6a_A12 Fwd TATGCTGCAGGGAGGGCCGACAACAAGTGCGAGAACAGCCTG(SEQ ID NO: 186) Ssm6a_A12 Rev GACTCCGGAGTTGGCGTGCAGCAGGTCCTGGCACTTCTTGAAG(SEQ ID NO: 187) Ssm6a_GGS_A12 TATGCTGCAGGGAGGGGCGGTAGCGCCGACAACAAGTGCGAGAACAG Fwd CCTG(SEQ ID NO: 188) Ssm6a_GGS_A12 ACTCCGGAGTTGGCACCGCCGCTGTGCAGCAGGTCCTGGCACTTCTTG Rev AAG(SEQ ID NO: 189) Kalio_A12_Fwd_B ATCTATGCTGCAGGGAGGGGTGTGGAAATTAACGTGAAGTGT(SEQ ID NO: 190) Kalio_A12 Rev GAGACTCCGGAGTTGGCCTTCGGCGTGCAGTGGCATTTACG(SEQ ID NO: 191) Moka1 A12 Fwd TATGCTGCAGGGAGGATCAACGTGAAGTGCAGCCTGCCCCAG(SEQ ID NO: 192) Moka1_A12 Rev GACTCCGGAGTTGGCGCTGTAGCACCGGCATTTCTTGTTCAT(SEQ ID NO: 193) Shk_A12 Fwd TATGCTGCAGGGAGGAGAAGCTGCATCGACACCATCCCCAAG(SEQ ID NO: 194) Shk_A12 rev GACTCCGGAGTTGGCACAGGTGCCGCAGGTCTTTCTACAGAAG(SEQ ID NO: 195) PcTx-1 A12 Fwd TATGCTGCAGGGAGGGAGGACTGCATCCCCAAGTGGAAGGGC(SEQ ID NO: 196) PcTX-1 A12 Rev GACTCCGGAGTTGGCGGTCTTAGGGGTCTTGGGCACGCACAC(SEQ ID NO: 197)

TABLE 10 KnotBody yield after the Protein-A purification of Toxin-antibody fusions. IgG1 Yield Toxin/Knottin Antibody (mg/L) Huwentoxin-IV KB_A07 6.0 ProTx-II 1.3 Ssm6a 11.6 Ssm6a_GGS 9.8 PcTx-I 11.5 Shk 9.4 Moka-1 3.7 Kaliotoxin (KTX) 5.0 Huwentoxin-IV KB_A12 10.7 ProTx-II 5.1 Ssm6a 7.4 Ssm6a_GGS 6.2 PcTx-I 11.9 Shk 11.8 Moka-1 3.9 Kaliotoxin (KTX) 5.3 Mambalgin-1 (Mba-1) 14.4 Mambalgin-2 (Mba-1) 7.8 EETI-II (Control) KB_A07 9.4 EETI-II (Control) KB_A12 11.8

TABLE 11A Summary IC₅₀ data for eight samples tested against Kv1.3 channels. IC₅₀ Sample type Toxin Antibody (nM) Toxin fusion Shk KB_A07 40 MoKa-1 N/E* Kaliotoxin (KTX) 900  Shk KB_A12 20 MoKa-1 N/E* Kaliotoxin (KTX) N/E* Isotype control None KB_A07 N/E* None KB_A12 N/E* N/E* = no detectable effect.

TABLE 11B Percentage ASIC1a current remaining in response to KB_A12 and KB_A07 based KnotBody applications followed by a full blocking concentration of PcTx1 % current remaining KnotBody/ Concentration % current PcTx1 Antibody (μM) remaining (300 nM) n KB_A07_PcTx1 6.6 6.2 3.6 5 KB_A12_PcTx1 5 4.8 3.8 7 KB_A12_Mba-1 3 69.2 2 5 KB_A12_Mba-2 2.3 72.4 1.9 5 KB_A07 (control) 6 70.9 1.9 3 KB_A12 (control) 3.3 67.4 2.7 6 Buffer only (control) 0 74.9 3.8 6

TABLE 12 Amino acid sequences of the LOX-1 binding adhirons. Adhiron Protein Sequence Ad-LOX1-A ATGVRAVPGNENSLEIEELARFAVDEHNKKENALLEFVRVV KAKEQTAHLDPLMDTMYYLTLEAKDGGKKKLYEAKVWVKF WMISDLIFNFKELQEFKPVGDA (SEQ ID NO: 26) Ad-LOX1-B ATGVRAVPGNENSLEIEELARFAVDEHNKKENALLEFVRVV KAKEQEQPIGEHPVNDTMYYLTLEAKDGGKKKLYEAKVWV KRWLRFTEIYNFKELQEFKPVGDA (SEQ ID NO: 27)

TABLE 13 Nucleotide sequences of the two adhirons used for this work (SEQ ID NOS: 198, 199) Adhiron gene*sequence Ad#LOX1#A GCTACAGGCGTGCGGGCTGTGCCCGGCAATGAGAACAGCCT GGAAATCGAGGAACTGGCCAGATTCGCCGTGGACGAGCACA ACAAGAAAGAGAACGCCCTGCTGGAATTCGTGCGGGTCGTG AAGGCCAAAGAGCAGTGGAGCGAGGCCGACAACGACTGGCA CACCATGTACTACCTGACCCTGGAAGCCAAGGACGGCGGCA AGAAGAAGCTGTACGAGGCCAAAGTGTGGGTCAAGCTGGAC CTGGAAACCTGGCAGCACTTCAACTTCAAAGAGCTCCAGGA ATTCAAGCCCGTGGGCGACGCT Ad#LOX1#B GCTACAGGCGTGCGGGCTGTGCCCGGCAATGAGAACAGCCT GGAAATCGAGGAACTGGCCAGATTCGCCGTGGACGAGCACA ACAAGAAAGAGAACGCCCTGCTGGAATTCGTGCGGGTCGTG AAGGCCAAAGAGCAGGAACAGCCCATCGGCGAGCACCCCGT GAACGACACCATGTACTACCTGACCCTGGAAGCCAAGGACG GCGGCAAGAAGAAGCTGTACGAGGCCAAAGTGTGGGTCAAG CGGTGGCTGCGGTTCACCGAGATCTACAACTTCAAAGAGCT CCAGGAATTCAAGCCCGTGGGCGACGCT

TABLE 14 sequences of the primers used for cloning adhirons into the CDR2 position of antibodies KB_A07 (SEQ ID NOS: 200 & 201) and KB_A12 (SEQ ID NOS: 202 & 203). Antibody Primer-name Sequence KB_A07 Ad_SUMO_10*A07*Fwd ATCTATGCTGCAGGGGTCGCTACAGGCGTGCGGGCTGTGCCCGGC Ad*SUMO10_A07*Rev GGGACTCCGGAGCGGCTTGCGTCGCCCACGGGCTTGAATTCCTGG KB_A12 Ad_SUMO_10*A12*Fwd ATCTATGCTGCAGGGAGGGCTACAGGCGTGCGGGCTGTGCCCGGC Ad*SUMO10_*A12*Rev GAGACTCCGGAGTTGGCGGCGTCGCCCACGGGCTTGAATTCCTGG

TABLE 15 N and C terminal linker sequences of “selected” KnotBodies and their variants. KnotBody N-terminal C-terminal construct linker sequence linker sequence KB_A07 (Parent) GV SR KB_A07 Gly4Ser GGGGS GGGGS (SEQ ID NO: 1) (SEQ ID NO: 1) KB_A07 (Gly4Ser)2 GGGGSGGGGS GGGGSGGGGS (SEQ ID NO: 204) (SEQ ID NO: 204) KB_A12 (Parent) GR AN KB_A12 Gly4Ser GGGGS GGGGS (SEQ ID NO: 1) (SEQ ID NO: 1) KB_A12 (Gly4Ser)2 GGGGSGGGGS GGGGSGGGGS (SEQ ID NO: 204) (SEQ ID NO: 204)

TABLE 16 Sequences of KnotBody linker variants. KnotBody construct Sequence KB_A07 GGTGGCGCTAGCGACATACAAATGACCCAATCACCTAGCTCTCTTAGTGCCTCTGTTGGGGATCGG GTCACCATCACTTGTAGAGCGAGCCAGAGTATCTCATCATACTTGAACTGGTACCAGCAGAAGCCAG GGAAGGCCCCCAAGCTGTTGATTTACGCGGCTGGGGTCTGCCCGCGCATCTTGATGAGGTGCAAA CAAGACTCAGACTGCCTGGCTGGATGTGTTTGCGGACCAAATGGTTTCTGCGGAAGCCGCTCAGGC GTGCCATCAAGATTTAGTGGTTCAGGAAGTGGTACGGACTTCACGCTGACGATTTCATCTCTTCAAC CCGAAGATTTCGCCACGTACTACTGTCAACAGGGTGCTTCTCCACCTTATACTTTCGGTCAGGGTAC CAAGGTTGAGATTAAGCGCACCGCGGCCGCAATC (SEQ ID NO: 205) KB_A07 Gly₄Ser GGTGGCGCTAGCGACATACAAATGACCCAAAGTCCGAGCTCCTTGAGTGCCTCCGTAGGTGATAGG GTCACTATTACTTGCAGAGCGTCTCAGTCCATCTCCTCCTATTTGAATTGGTACCAACAGAAACCGG GGAAAGCCCCTAAGCTCCTGATCTACGCCGCTGGGGGAGGCGGGAGTGGGGGGGGCGGGTCCTG TCCGCGCATCCTTATGCGGTGTAAACAGGACAGTGATTGCCTTGCTGGTTGTGTCTGCGGCCCCAA TGGTTTTTGCGGGGGTGGGGGGGGCAGCGGTGGGGGCGGTTCCTCCGGGGTGCCATCTCGCTTT AGCGGTTCAGGTAGTGGAACGGACTTTACACTGACAATATCATCTTTGCAACCAGAGGATTTCGCCA CGTACTACTGTCAGCAAGGTGCCTCTCCACCTTACACGTTTGGACAAGGCACCAAAGTAGAGATTAA GCGGACCGCGGCCGCAATC (SEQ ID NO: 206) KB_A07 (Gly₄Ser)₂ GGTGGCGCTAGCGACATACAAATGACCCAATCACCTAGCTCTCTTAGTGCCTCTGTTGGGGATCGG GTCACCATCACTTGTAGAGCGAGCCAGAGTATCTCATCATACTTGAACTGGTACCAGCAGAAGCCA GGGAAGGCCCCCAAGCTGTTGATTTACGCGGCTGGCGGAGGAGGGTCCTGCCCGCGCATCTTGAT GAGGTGCAAACAAGACTCAGACTGCCTGGCTGGATGTGTTTGCGGACCAAATGGTTTCTGCGGAGG AGGCGGAGGTTCCTCAGGCGTGCCATCAAGATTTAGTGGTTCAGGAAGTGGTACGGACTTCACGCT GACGATTTCATCTCTTCAACCCGAAGATTTCGCCACGTACTACTGTCAACAGGGTGCTTCTCCACCT TATACTTTCGGTCAGGGTACCAAGGTTGAGATTAAGCGCACCGCGGCCGCAATC (SEQ ID NO: 207) KB_A12 GGTGGCGCTAGCCAGAGTGTCTTGACGCAGCCACCTTCTGTCAGCGAGGCCCCACGCCAGAGGGT TACCATAACATGTTCCGGGTCCAGCTCTAACATAGGGAATAACGCGGTAAACTGGTATCAGCAATTG CCCGGCAAAGCACCGAAACTCTTGATCTATGCAGCGGGGAGGTGTCCTCGAATACTGATGCGATGT AAACAGGACTCCGATTGTCTTGCGGGATGTGTGTGTGGTCCGAATGGGTTTTGCGGCGCCAACAGC GGCGTAAGTGATCGATTCTCAGCGGCGAAATCCGGCACATCCGCCTCACTGGCGATCAACGGATTG CGAAGTGAGGACGAAGCTGACTATTATTGCGCGGCCTGGGATGATTCCTTGAACGGGTATGTATTT GGCACAGGAACGAAGCTGACTGTGCTGGGACAACCCGCGGCCGCAATC (SEQ ID NO: 208) KB_A12 Gly₄Ser GGTGGCGCTAGCCAGAGCGTACTGACGCAGCCGCCTTCTGTTAGCGAGGCTCCCCGACAGCGAGT AACGATAACGTGCAGCGGTTCAAGCAGTAATATCGGGAATAATGCAGTAAATTGGTATCAGCAACTG CCTGGAAAAGCGCCCAAGCTGCTCATATATGCGGCCGGGGGCGGGGGTAGCGGCGGAGGGGGAA GCTGCCCAAGAATCTTGATGCGGTGTAAACAAGATTCAGACTGTTTGGCCGGTTGCGTATGCGGTC CAAATGGGTTCTGCGGAGGTGGTGGTGGGTCCGGTGGAGGAGGTAGTAGCGGGGTTAGTGATCGA TTCTCCGCGGCGAAGTCCGGCACCAGTGCAAGTCTCGCTATAAACGGGCTCAGGTCAGAAGATGA GGCAGATTATTACTGTGCCGCATGGGACGACAGTTTGAACGGCTATGTCTTCGGAACGGGGACTAA ACTTACCGTACTTGGACAGCCCGCGGCCGCAATC (SEQ ID NO: 209) KB_A12 (Gly₄Ser)₂ GGTGGCGCTAGCCAGAGTGTCTTGACGCAGCCACCTTCTGTCAGCGAGGCCCCACGCCAGAGGGT TACCATAACATGTTCCGGGTCCAGCTCTAACATAGGGAATAACGCGGTAAACTGGTATCAGCAATTG CCCGGCAAAGCACCGAAACTCTTGATCTATGCAGCGGGAGGGGGAGGCTCTTGTCCTCGAATACT GATGCGATGTAAACAGGACTCCGATTGTCTTGCGGGATGTGTGTGTGGTCCGAATGGGTTTTGCGG CGGTGGGGGCGGCTCTAGCGGCGTAAGTGATCGATTCTCAGCGGCGAAATCCGGCACATCCGCCT CACTGGCGATCAACGGATTGCGAAGTGAGGACGAAGCTGACTATTATTGCGCGGCCTGGGATGATT CCTTGAACGGGTATGTATTTGGCACAGGAACGAAGCTGACTGTGCTGGGACAACCCGCGGCCGCA ATC (SEQ ID NO: 210)

TABLE 17A Loop1 sequences of unique cMET binders isolated from the KnotBody loop library. Cysteines flanking the loop 1 residues are highlighted in bold. TRF Clone ID Loop 1 sequence signal KB_A12_cMET_H01 CHRMSGTGRC (SEQ ID NO: 211) 131578 KB_A12_cMET_F05 CKRLMSGAGRC 59604 (SEQ ID NO: 212) KB_A12_cMET_G01 CQRQSGTGRC (SEQ ID NO: 213) 98924 KB_A12_cMET_G07 CRASSGTGRC (SEQ ID NO: 214) 57958 KB_A12_cMET_E07 CPKRHTGTGRC 95275 (SEQ ID NO: 215) KB_A12_cMET_E04 CGHLSGTGRC (SEQ ID NO: 216) 41255 KB_A12_cMET_F02 CGRASGTGRC (SEQ ID NO: 217) 98503 KB_A12_cMET_H05 CNRASGAGRC (SEQ ID NO: 218) 152852 KB_A12_cMET_H03 CRGMTGVGRC (SEQ ID NO: 219) 54161 KB_A12_cMET_H08 CQTGRSGTGRC 6730 (SEQ ID NO: 220) KB_A12_cMET_E05 CDRKAGTGRC (SEQ ID NO: 221) 115765 KB_A12_cMET_E11 CAKKSGTGRC (SEQ ID NO: 222) 158497 KB_A12_cMET_E12 CAKRSGTGRC (SEQ ID NO: 223) 95895 KB_A12_cMET_H04 CPQMTGTGRC (SEQ ID NO: 224) 112989 KB_A12_cMET_E01 CNLTSGTGRC (SEQ ID NO: 225) 22439 KB_A12_cMET_G10 CEKHSGTGRC (SEQ ID NO: 226) 64760 KB_A12_cMET_H02 CAKRTGTGRC (SEQ ID NO: 227) 35448 KB_A12_cMET_F06 CPHQTGTGRC (SEQ ID NO: 228) 56209 KB_A12_cMET_F10 CRSLMSGTGRC 51323 (SEQ ID NO: 229) KB_A12_cMET_F12 CKAQSGSGRC (SEQ ID NO: 230) 119476 KB_A12_cMET_G06 CNKSGGTGRC (SEQ ID NO: 231) 62077 KB_A12_cMET_E02 CRKKSGANRC (SEQ ID NO: 232) 9482 KB_A12_cMET_G08 CARLSGSGRC (SEQ ID NO: 233) 27930

TABLE 17B Loop1 sequences of unique β-galactosidase binders isolated from the KnotBody loop library. Cysteines flanking the loop 1 residues are highlighted in bold. KB_A12_B-gal_F02 CRTTSTIRRRC 476399 (SEQ ID NO: 234) KB_A12_B-gal_G05 CLDTVNLRRRC 529017 (SEQ ID NO: 235) KB_A12_B-gal_H03 CNQTMSIRRPC 517907 (SEQ ID NO: 236) KB_A12_B-gal_E04 CLATTSIRRPC 523482 (SEQ ID NO: 237) KB_A12_B-gal_E05 CRETSSLRRPC 311927 (SEQ ID NO: 238) KB_A12_B-gal_F09 CIATSHIRRHC 257676 (SEQ ID NO: 239) KB_A12_B-gal_F11 CHETAQIRRRC 531474 (SEQ ID NO: 240) KB_A12_B-gal_H07 CTQTALIRRRC 62049 (SEQ ID NO: 241) KB_A12_B-gal_H02 CEQTSVIRRHC 501461 (SEQ ID NO: 242) KB_A12_B-gal_F03 CLATTSIRRHC 288901 (SEQ ID NO: 243) KB_A12_B-gal_F07 CRTTANVRRHC 246274 (SEQ ID NO: 244)

TABLE 18 Nucleotide sequences of mutagenic oligos used for constructing KnotBody loop library. Mutagenic oligo Nucleotide sequence A12_L1 (6) ACAGGCAGTCTGAGTCCTGTTTGCASNBSNBSNBSNBS NBSNBGCACCTCCCTGCAGCATAGATCA G_((SEQ ID NO: 245)) A12_L1 (8) CAGGCAGTCTGAGTCCTGTTTGCASNBSNBSNBSNBSN BSNBSNBSNBGCACCTCCCTGCAGCATAGATCA G_((SEQ ID NO: 246)) A12_L1 (9) CAGGCAGTCTGAGTCCTGTTTGCASNBSNBSNBSNBSN BSNBSNBSNBSNBGCACCTCCCTGCAGCATAGATCA G_((SEQ ID NO: 247)) A12_L1 (10) CAGGCAGTCTGAGTCCTGTTTGCASNBSNBSNBSNBSN BSNBSNBSNBSNBSNBGCACCTCCCTGCAGCATAGATCA G_((SEQ ID NO: 248))

TABLE 19 Percentage transcytosis of 21 test KnotBodies and controls across the BBB in an in vitro model system. Test well Clones % transcytosis after 6 h C1 KB_TFR_B01 0.0 KB_TFR_B02 KB_TFR_B03 C2 KB_TFR_B05 1.3 KB_TFR_B07 KB_TFR_B09 C3 KB_TFR_B11 0.9 KB_TFR_C01 KB_TFR_C03 C4 KB_TFR_C05 0.7 KB_TFR_C06 KB_TFR_C07 C5 KB_TFR_D02 0.0 KB_TFR_D05 KB_TFR_D07 C6 KB_TFR_D10 0.1 KB_TFR_E03 KB_TFR_E06 B1 KB_TFR_A05 0.0 KB_TFR_A06 KB_TFR_A07 OX26-Fab Positive Control 0.2 KB_A12 Negative control 0.1

TABLE 20 Amino acid sequences of anti-TFR VHs that could potentially cross BBB. Name Sequence KB_TFR_B05 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGRNIPI LGIANYAQKSRGRVTITADESTSTAYMELSSLRSEDTAVYYCARVAPYSSGWANVD AFDIWGQGTLVTVSS_((SEQ ID NO: 249)) KB_TFR_B07 QVQLVQSEPEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGRIIPIF GIANYAQKFQGRVTITADKSTSTAYMELSSLRSEDTAVYYCAGITGTNGPHDAFDI WGQGTMVTVSS_((SEQ ID NO: 250)) KB_TFR_B09 QVQLVQSGAEVKKPGSSVKVSCKASGGTFSSYTISWVRQAPGQGLEWMGRIIPIL SIANYAQKFQSRVTITADESTSTAYMELSSLRSEDTAVYYCARVRPYYDSSADLDAF DIWGQGTMVTVSS_((SEQ ID NO: 251)) KB_TFR_B11 EVQLVQFGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGRIN PNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSEDTAVYYCARGRGPLSYW GQGTLVTVSS_((SEQ ID NO: 252)) KB_TFR_C01 QVQLVQSGAEVKKPGSSVKVSCKASGGTYSSNTFTWVRQAPGQGLEWMGRIIP VLDLTNSAVNFQDRVTITADESTSTVYMELSSLRSEDTAVYYCASTTAMVPTDAFD IWGQGTMVTVSS_((SEQ ID NO: 253)) KB_TFR_C03 EVQLVESGAEVKKPGSSVKVSCKASGGTVSSYAISWVRQAPGQGLEWMGGIIPIF GTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCARDLFQRSGSYPYYY YYYGMDVWGQGTMVTVSS_((SEQ ID NO: 254)) KB_TFR_C05 QVQLVQSGAEVKKPGSSVKVSCKASGGTYSSNTFTWVRQAPGQGLEWMGRIIPI LGIANYAQKFQGRVTITADESTSTAYMVLSSLRSEDTAVYYCARVRPYYDSSADLD AFDIWGQGTLVTVSS_((SEQ ID NO: 255)) KB_TFR_C06 QVQLVESGAEVKKPGSSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMGGIIPIF GTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAVYYCAIALRDSSGYYTDAL DIWGQGTLVTVSS_((SEQ ID NO: 256)) KB_TFR_C07 QMQLVQSGAEVKKPGASVKVSCKASGYTFTSYGISWVRQAPGQGLEWMGGIIPI FGTANYAQKFQGRVTITADESTGTAYMELSSLRSEDTAVYYCAGPGGYYDILTGYP TDAFDIWGQGTMVTVSS_((SEQ ID NO: 257))

TABLE 21 Nucleotide sequences of EETI-II-Cow ULVC-Ab synthetic gene. Name Sequence EETI-II Cow CAGCCGGCCATGGCCCAAGTGCAACTTCGCGAAAGCGGGCCTTCCTTGGTTA ULVC-Ab AACCATCGCAGACCCTGAGTCTTACGTGCACGGCCAGCGGTTTTTCCCTGTC CGATAAAGCAGTAGGGTGGGTGCGCCAAGCGCCGGGAAAGGCATTAGAGTG GCTTGGTTCCATTGATACGGGGGGGAACACAGGATACAATCCCGGCTTGAAG TCTCGCCTTTCAATCACCAAAGACAACTCAAAGTCTCAAGTCTCCCTGAGCGT CTCATCGGTTACCACCGAGGACTCAGCCACGTACTACTGCACATCCGTTCATC AGGAGACCAAAAAATACCAATCTTGTCCCCGCATCCTGATGCGTTGTAAACAA GACAGCGACTGCCTGGCAGGTTGTGTATGCGGTCCTAATGGGTTTTGTGGAT TAACCACTCTGCCAGTCTCATATTCTTACACTTATAATTATGAGTGGCATGTTG ATGTCTGGGGGCAGGGGCTTCTTGTCACGGTCTCGAGTGGTGGAGGCGGTT CAGGCGGAGGTGGCTCTGGCGGTGGCGCTAGCCAGGCTGTTTTAAACCAAC CCAGCTCGGTTAGTGGATCGTTGGGGCAGCGCGTCTCAATCACTTGTTCCGG CTCCTCCTCGAATGTTGGTAATGGATATGTTTCCTGGTACCAGCTTATCCCCG GTAGCGCTCCTCGCACCTTAATTTATGGTGACACGTCCCGTGCCAGCGGTGT CCCTGATCGTTTCTCTGGGAGCCGTTCCGGGAACACCGCCACACTTACAATT AGTAGCCTTCAAGCTGAGGACGAAGCGGACTATTTTTGTGCCTCTGCAGAGG ATTCATCCTCTAACGCTGTGTTTGGCAGTGGCACCACCCTTACTGTGTTAGGG GCGGCCGCAGAT (SEQ ID NO: 258)

TABLE 22 Primers used to create inserts for the cloning of KnotBodies and linker libraries for mammalian display. Primer Sequence 2979 CTAAGCTCCTGATCTATGCTGCAGGGGTCTGCCCGCGTATCCTG ATGCGTTGCAAACAGGACTCAGACTGCCTG G_((SEQ ID NO: 259)) 2980 GAACCTTGATGGGACTCCGGAGCGGCTCCCGCAGAAGCCGTTCG GACCGCATACGCAGCCGGCCAGGCAGTCTGAGTCCT G_((SEQ ID NO: 260)) 2983 CTAAGCTCCTGATCTATGCTGCAGGGAGGTGCCCGCGTATCCTG ATGCGTTGCAAACAGGACTCAGACTGCCTG G_((SEQ ID NO: 261)) 2984 GAATCGGTCAGAGACTCCGGAGTTGGCCCCGCAGAAGCCGTTCG GACCGCATACGCAGCCGGCCAGGCAGTCTGAGTCCT G_((SEQ ID NO: 262)) 2985 TTTTTTCCATGGCTGAAGTCCAACTG_((SEQ ID NO: 263)) 2995 TTTTTTGCGGCCGCGGTACGTTTG_((SEQ ID NO: 264)) 2999 TGCAGCATAGATCAGGAGCTTAG_((SEQ ID NO: 265)) 3000 TCCGGAGTCCCATCAAGGTTC_((SEQ ID NO: 266)) 3002 TTTTTTGCGGCCGCAGGCTGACCTAG_((SEQ ID NO: 267)) 3003 TGCAGCATAGATCAGGAGCTTAG_((SEQ ID NO: 268)) 3004 TCCGGAGTCTCTGACCGATTC_((SEQ ID NO: 269))

TABLE 23 Amino acid sequence of the parental anti-TACE D1A12 scFv¹⁰¹  used for knottin linker library insertion into VH and VL CDRs 1 to 3. EVQLVESGGGLVRPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLE WVSAISGSGGSTYYADSVKGRFTISRDNTKNSLYLQMTSLRADDTAFYY CVDFGPGYGTGWFDYWGPGTLVTVSSGGGGSGGGGSGGGASDIQMT QSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIHDASSL QSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQSFSIPLTFGGGTK MDIKRT (SEQ ID NO: 270)

TABLE 24 Primers used to create inserts for the cloning of KnotBodies and linker libraries for phage display selection. Primer Sequence 2965 GCGTCGGGTTTTACCTTTAGCVNSVNSTGTCCCCGGATACTTATGAGATGTAAGCAA GATAGTGATTGCCTCGC (SEQ ID NO: 271) 2966 CGGAGCCTGACGAACCCASNBSNBGCCACAAAACCCGTTGGGACCACAGACACAT CCGGCGAGGCAATCACTATCTTGC (SEQ ID NO: 272) 2967 GTAAAGGCCTGGAATGGGTCTCCVNSVNSTGTCCCCGGATACTTATGAGATGTAAG CAAGATAGTGATTGCCTCGC (SEQ ID NO: 273) 2968 GTCGCGAGAGATGGTGAAACGSNBSNBGCCACAAAACCCGTTGGGACCACAGACA CATCCGGCGAGGCAATCACTATCTTGC (SEQ ID NO: 274) 2969 CACGGCTTTTTATTACTGCGTTGATVNSVNSTGTCCCCGGATACTTATGAGATGTAA GCAAGATAGTGATTGCCTCGC (SEQ ID NO: 275) 2970 CAGGGTGCCCGGACCCCASNBSNBGCCACAAAACCCGTTGGGACCACAGACACAT CCGGCGAGGCAATCACTATCTTGC (SEQ ID NO: 276) 2971 GAGACAGAGTCACCATCACTTGCVNSVNSTGTCCCCGGATACTTATGAGATGTAAG CAAGATAGTGATTGCCTCGC (SEQ ID NO: 277) 2972 CCTGGCTTCTGCTGATACCASNBSNBGCCACAAAACCCGTTGGGACCACAGACACA TCCGGCGAGGCAATCACTATCTTGC (SEQ ID NO: 278) 2973 CCCTAAGCTCCTGATCCATGATVNSVNSTGTCCCCGGATACTTATGAGATGTAAGCA AGATAGTGATTGCCTCGC (SEQ ID NO: 279) 2974 CTGAACCTTGATGGGACCCCSNBSNBGCCACAAAACCCGTTGGGACCACAGACAC ATCCGGCGAGGCAATCACTATCTTGC (SEQ ID NO: 280) 2975 CCTGAAGATTTTGCAACTTACTACTGTVNSVNSTGTCCCCGGATACTTATGAGATGT AAGCAAGATAGTGATTGCCTCGC (SEQ ID NO: 281) 2976 CATTTTGGTCCCTCCGCCGAASNBSNBGCCACAAAACCCGTTGGGACCACAGACAC ATCCGGCGAGGCAATCACTATCTTGC (SEQ ID NO: 282) 2977 CTAAGCTCCTGATCTATGCTGCAVNSVNSTGCCCGCGTATCCTGATGCGTTGCAAA CAGGACTCAGACTGCCTGG (SEQ ID NO: 283) 2978 GAACCTTGATGGGACTCCGGASNBSNBCCCGCAGAAGCCGTTCGGACCGCATACG CAGCCGGCCAGGCAGTCTGAGTCCTG (SEQ ID NO: 284) 2981 CTAAGCTCCTGATCTATGCTGCAVNSVNSTGCCCGCGTATCCTGATGCGTTGCAAA CAGGACTCAGACTGCCTGG (SEQ ID NO: 285) 2982 GAATCGGTCAGAGACTCCGGAGTTGGCCCCGCAGAAGCCGTTCGGACCGCATACG CAGCCGGCCAGGCAGTCTGAGTCCTG (SEQ ID NO: 286) 2986 GCTAAAGGTAAAACCCGACGC (SEQ ID NO: 287) 2987 TGGGTTCGTCAGGCTCCG (SEQ ID NO: 288) 2988 TTTTTTCTCGAGACGGTCACCAGG (SEQ ID NO: 289) 2989 GGAGACCCATTCCAGGCCTTTAC (SEQ ID NO: 290) 2990 CGTTTCACCATCTCTCGCGAC (SEQ ID NO: 291) 2991 ATCAACGCAGTAATAAAAAGCCGTG (SEQ ID NO: 292) 2992 TTTTTTGCTAGCGACATCCAGATGACC (SEQ ID NO: 293) 2993 GCAAGTGATGGTGACTCTGTCTC (SEQ ID NO: 294) 2994 TGGTATCAGCAGAAGCCAGG (SEQ ID NO: 295) 2996 ATCATGGATCAGGAGCTTAGGG (SEQ ID NO: 296) 2997 GGGGTCCCATCAAGGTTCAG (SEQ ID NO: 297) 2998 ACAGTAGTAAGTTGCAAAATCTTCAGG (SEQ ID NO: 298) 3001 TTTTTTGCTAGCCAGTCTGTGCTGAC (SEQ ID NO: 299) 3005 AAATTTACTCGAGACGGTCACCAGGGTGCCCGGACCCCA (SEQ ID NO: 300) 3006 TTTTTTGCGGCCGCGGTACGTTTGATATCCATTTTGGTCCCTCCGCCGAA (SEQ ID NO: 300) 3012 TGGGGTCCGGGCACCCTG (SEQ ID NO: 301) D1A12 TTCTGCGGCCGCGGTACGTTTGATATCCATT (SEQ ID NO: 78) NotI Rev

TABLE 25 Primers pairs required to create the DNA inserts needed for the three fragment assembly to create the knottin linker libraries. The VL CDR3 knottin linker library was created by a 2 fragment assembly with inserts F1 and F2. CDR Primer Template PCR size Library insertion Insert pairs scFv (bp) A VH CDR1 A1 2985/2986 D1A12 98 A2 2965/2966 None 132 A3 2987/2995 D1A12 638 B VH CDR2 B1 2985/2989 D1A12 155 B2 2967/2968 None 137 B3 2990/2995 D1A12 545 C VH CDR3 C1 2985/2991 D1A12 302 C2 2969/2970 None 136 C3 3012/2995 D1A12 413 D VL CDR1 D1 2985/2993 D1A12 485 D2 2971/2972 None 136 D3 2994/2995 D1A12 233 E VL CDR2 E1 2985/2996 D1A12 566 E2 2973/2974 None 135 E3 2997/2995 D1A12 167 F VL CDR3 F1 2985/2998 D1A12 680 F2 2975/2976 None 141 N/A G KB_A07 G1 2985/2999 A07-knotbody 569 G2 2977/2978 None 137 G3 3000/2995 A07-knotbody 170

TABLE 26 Sequences of linkers capable of functional knottin display embedded in D1A12 VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 and VL CDR3. XX represents the randomised residues indicated in FIG. 28. Clone CDR insertion N-term XX C-term XX A_A01 VH CDR1 AV TK A_A02 VH CDR1 KM SD A_A03 VH CDR1 PI LP A_A04 VH CDR1 EK PR B_A05 VH CDR2 GK TP B_A06 VH CDR2 RL ED B_A07 VH CDR2 PK GN B_A08 VH CDR2 RP TP B_A10 VH CDR2 VP SL B_A11 VH CDR2 KG TN B_A12 VH CDR2 GT SD C_B01 VH CDR3 KP TK C_B02 VH CDR3 RP SQ C_B03 VH CDR3 PT TV C_B04 VH CDR3 KG QG C_B05 VH CDR3 RP GP C_B06 VH CDR3 KG QG C_B07 VH CDR3 RP DN C_B08 VH CDR3 KG QG D_B09 VL CDR1 RG QD D_B10 VL CDR1 IS HM E_B11 VL CDR2 RA AQ E_B12 VL CDR2 RK SR E_C01 VL CDR2 QP TR E_C02 VL CDR2 RP SR E_C03 VL CDR2 RK VN E_C04 VL CDR2 KA HQ E_C05 VL CDR2 VP HT E_C06 VL CDR2 EK TP E_C08 VL CDR2 KV TE E_C09 VL CDR2 RR GE E_C10 VL CDR2 KP AN E_C11 VL CDR2 LP SI E_C12 VL CDR2 HP SS E_D01 VL CDR2 KP AT E_D02 VL CDR2 AP VD E_D03 VL CDR2 LK SP E_D04 VL CDR2 HR DT E_D05 VL CDR2 KG TN E_D06 VL CDR2 RP DH E_D07 VL CDR2 KP HK E_D08 VL CDR2 QP HE E_D09 VL CDR2 TK AR E_D10 VL CDR2 VK NP E_D11 VL CDR2 TK TT E_D12 VL CDR2 KP AN E_E01 VL CDR2 TK DT E_E02 VL CDR2 QP TT E_E03 VL CDR2 RV DD E_E04 VL CDR2 RK SN E_E05 VL CDR2 RG HS E_E06 VL CDR2 QP SH E_E07 VL CDR2 AK TR E_E08 VL CDR2 RP TS F_E09 VL CDR3 RG QH F_E10 VL CDR3 RG ND F_E11 VL CDR3 PK TR F_E12 VL CDR3 KP GQ F_F01 VL CDR3 QI SR F_F02 VL CDR3 EK SN F_F03 VL CDR3 RG TH F_F04 VL CDR3 EK TH F_F05 VL CDR3 PI SE F_F06 VL CDR3 KG QH F_F07 VL CDR3 KG HE

TABLE 27 Sequences of linkers capable of functional knottin display embedded in KB_A07 VL CDR2. XX represents the randomised residues indicated in FIG. 28. CDR N-term C-term DELFIA Clone insertion linker linker signal G_F08 VL CDR2 EK AH 15940 G_F09 VL CDR2 PR AS 13431 G_F10 VL CDR2 PP TK 11667 G_F11 VL CDR2 HK SR 14160 G_F12 VL CDR2 GK SR 11209 G_G01 VL CDR2 QK TV 10863 G_G03 VL CDR2 TP ST 14493 G_G04 VL CDR2 LP AR 14330 G_G05 VL CDR2 GV GE 10429 G_G06 VL CDR2 NK AP 10255 G_G07 VL CDR2 KP TR 26510 G_G08 VL CDR2 KP HR 13848 G_G09 VL CDR2 KP TE 24701 G_G10 VL CDR2 RP NS 12258 G_G11 VL CDR2 PR SD 15883 G_G12 VL CDR2 PR TS 14911 G_H01 VL CDR2 TK AA 10714 G_H02 VL CDR2 RR TR 11930 G_H03 VL CDR2 RV SG 12457 G_H04 VL CDR2 RR TE 20675 G_H05 VL CDR2 TR TT 10654 G_H06 VL CDR2 PG TL 17126

TABLE 28 Alternative framings of EETI-II donor Name of format Sequence SEQ ID EET 1-5 CPRILMRCKQDSDCLAGCVCGPNGFC 302 EET 2-6 CKQDSDCLAGCVCGPNGFCGSGSDGGVC 303 EET 3-1 CLAGCVCGPNGFCGSGSDGGVCPRILMRC 304 EET 4-2 CVCGPNGFCGSGSDGGVCPRILMRCKQDSDC 305 EET 5-3 CGPNGFCGSGSDGGVCPRILMRCKQDSDCLAGC 306 EET 6-4 CGSGSDGGVCPRILMRCKQDSDCLAGCVC 307

TABLE 29 Sequence of KnotBodies with alternative framing of EETI_II C09 (9820) (SEQ ID NO: 308) QSVLTQPPSVSEAPRQRVTITCggkr CGPNGFCGSGSDGGVCPRILMRCK QDSDCLAGC tsNWYQQKPGQAPVLVVQDDSVRPSGIPERFSGSNSGNTAT LTISRVEAGDGADYYCQVWDISSDLGVFGGGTKLTVLGQP E01 (127988) (SEQ ID NO: 309) QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIY aagg CGPNGFCGSGSDGGVCPRILMRCKQDSDCLAGC gihSGVSDRFSDS KSGTSASLAISGLQSEDEADYYCAAWDDSLNGYVFGTGTKLTVLGQP E08 (121047) (SEQ ID NO: 310) QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIY aagg CGPNGFCGSGSDGGVCPRILMRCKQDSDCLAGC giqSGIPERFSGS KSGTSASLAISGLRSEDEADYYCAAWDDNLSGYVFGTGTKLTVLGQP E05 (118631) (SEQ ID NO: 311) QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIY aaggCGPNGFCGSGSDGGVCPRILMRCKQDSDCLAGCgaqSGIPERFSGS KSGTSASLAISGLQSEDEAHYYCAAWDDSLNGYVFGTGTKVTVLGQP F01 (101798) (SEQ ID NO: 312) DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIDa age CGPNGFCGSGSDGGVCPRINMRCKQDSDCLAGC grdSGVPSRFSGSG SGTDFTLTISSLQPEDFATYYCQQSYSTPWTFGQGTKVEIKRT E03 (82495) (SEQ ID NO: 313) QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIY aaggCGPNGFCGSGSDGGVCPRILMRCKQDSDCLAGCgrnSGIPERFSGS KSGHTATLTISRVEAGDEADYYCQVWDSSSDHVLFGGGTKLTVLGQP F07 (72824) (SEQ ID NO: 314) QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIY aaggCGPNGFCGSGSDGGVCPRILMRCKQDSDCLAGCggqSGVPDRFSGS KSGTSASLAISRLQSEDEADYYCAAWDDSLNAYVFGTGTKVTVLGQP E09 (70274) (SEQ ID NO: 315) QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIY aaggCGPNGFCGSGSDGGVCPRILMRCKQDSDCLAGCgtqSGIPERFSGS KSGTSASLAISGLRSEDEADYYCAAWDDSLRAYVFGTGTQLTVLGQP F03 (67972) (SEQ ID NO: 316) QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGRAPKLLIY aaggCGPNGFCGSGGDGGVCPRILMRCKQDSDCLAGCgahSGIPERFSGS KSGTSASLAISGLRSEDEADYYCAAWDDSLNGWVFGGGTKVTVLGQP G11 (27650) (SEQ ID NO: 317) QSVLTQPPSVSEAPRQRVTITCSGSSSNIGNNAVNWYQQLPGKAPKLLIY aaggCGPNGFCGSGSDGGVCPRILMRCKQDSDCLAGCgqsSGVSDRFSGS KSGTSATLDITGLQTGDEADYYCGTWDNSLSVGVFGGGTKVTVLGQP

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1. A method of screening comprising: (i) providing a founder library of binding members, each binding member in the library comprising a fusion protein and optionally, a partner domain associated with the fusion protein, wherein the fusion protein comprises a donor diversity scaffold domain inserted within a recipient diversity scaffold domain, optionally wherein N and C terminals of the donor diversity scaffold domain are linked to the recipient diversity scaffold domain with linkers of up to 4 amino acids, wherein the donor diversity scaffold domain comprises a donor scaffold and a donor interaction sequence and the recipient diversity scaffold domain comprises a recipient scaffold and a recipient interaction sequence, and one, two or all three of the donor interaction sequence, the recipient interaction sequence and the linkers are diverse in said founder library, (ii) screening the founder library for binding members which display a binding activity, and (iii) identifying one or more binding members in the founder library which display the binding activity. 2-6. (canceled)
 7. A method according to claim 1, comprising further enriching the one or more binding members by: (a) recovering the one or more binding members, (b) subjecting the recovered binding members to selection for the binding activity, (c) recovering the one or more selected binding members (d) optionally repeating steps (b) and (c) one or more times.
 8. A method according to claim 1, further comprising: (iv) introducing diverse amino acid residues at one or more positions in the amino acid sequence of one or more identified founder binding members to produce a modified library of binding members, (v) screening the modified library for modified binding members which display a binding activity and (vi) identifying one or more modified binding members in the modified library which display the binding activity. 9-11. (canceled)
 12. The method according to claim 8, comprising further enriching the one or more binding members by: (e) recovering the one or more modified binding members, (f) subjecting the recovered modified binding members to selection for the binding activity, (g) recovering the one or more selected binding modified members and (h) optionally repeating steps (f) and (g) one or more times. 13-18. (canceled)
 19. The method according to claim 8, further comprising: (vii) associating the fusion protein from the one or more identified binding members or modified binding members with a diverse population of binding partners to produce a shuffled library of binding members, (viii) screening the shuffled library for shuffled binding members which display a binding activity, (ix) identifying one or more shuffled binding members which display the binding activity.
 20. The method according to claim 19, comprising further enriching the one or more shuffled binding members by: (i) recovering the one or more shuffled binding members, (j) subjecting the recovered shuffled binding members to selection for the binding activity, (k) recovering the one or more selected binding modified members and (l) optionally repeating steps (j) and (k) one or more times. 21-23. (canceled)
 24. The method according to claim 1, wherein both the donor and recipient diversity scaffolds of the fusion protein contribute to the binding activity of the fusion protein. 25-35. (canceled)
 36. The method according to claim 1, wherein the method further comprises isolating and/or purifying the one or more identified binding members from the library.
 37. The method according to claim 1, wherein the binding members in the library are displayed on the surface of a ribosome, cell or virus which comprises the nucleic acid encoding the binding member.
 38. The method according to claim 37, wherein the method comprises isolating and/or purifying the ribosome, cell or virus displaying the one or more identified binding members from the library.
 39. The method according to claim 37, wherein the method comprises isolating and/or purifying the nucleic acid encoding the one or more identified binding members from the library.
 40. The method according to claim 37, wherein the method comprises amplifying and/or cloning the nucleic acid encoding the one or more identified binding members from the library.
 41. (canceled)
 42. The method according to claim 37, further comprising synthesising or recombinantly expressing one or more identified binding members from the library.
 43. The method according to claim 1, further comprising determining the effect of the one or more binding members identified from the library on the activity of a target molecule.
 44. The method according to claim 43, wherein the target molecule is an ion channel and the effect of the one or more identified binding members on ion flow through the channel is determined
 45. The method according to claim 1, further comprising synthesising or recombinantly expressing an isolated donor diversity scaffold domain from one or more of the identified binding members from the founder library.
 46. The method according to claim 1, further comprising re-formatting one or more of the identified binding members as an scFv, Fab, scFv-Fc, Fc, IgA, IgD, IgM or IgG.
 47. The method according to claim 1, further comprising attaching a therapeutic moiety, half-life extension moiety or detectable label to the one or more identified binding members.
 48. (canceled)
 49. A method of producing a library of binding members comprising: providing a population of nucleic acids encoding a diverse population of fusion proteins comprising a donor diversity scaffold domain inserted into a recipient diversity scaffold domain, wherein the donor diversity scaffold domain comprises a donor scaffold and a donor interaction sequence and the recipient diversity scaffold domain comprises a recipient scaffold and a recipient interaction sequence, optionally wherein the N and C terminals of the donor diversity scaffold domain are linked to the recipient diversity scaffold domain with linkers of up to 4 amino acids, and one, two or all three of the donor interaction sequence, the recipient interaction sequence and the linkers are diverse in said population, expressing said population of nucleic acids to produce the diverse population, and optionally associating the fusion proteins with a population of partner domains, thereby producing a library of binding members.
 50. The method according to claim 49, wherein the nucleic acids are expressed in a cell or cell-free ribosome.
 51. The method according to claim 50, wherein the cell is a prokaryotic cell.
 52. The method according to claim 50, wherein the cell is a eukaryotic cell.
 53. The method according to claim 52, wherein the eukaryotic cell is a plant, yeast, insect or mammalian cell.
 54. A library of binding members comprising: a diverse population of fusion proteins comprising a donor diversity scaffold domain inserted into a recipient diversity scaffold domain, wherein the donor diversity scaffold domain comprises a donor scaffold and a donor interaction sequence and the recipient diversity scaffold domain comprises a recipient scaffold and a recipient interaction sequence, optionally wherein the N and C terminals of the donor diversity scaffold domain are linked to the recipient diversity scaffold domain with linkers of up to 4 amino acids, and one, two or all three the donor interaction sequence, the recipient interaction sequence and the linkers are diverse in said population, and optionally wherein the fusion proteins are associated with binding partners to form heterodimers. 55-62. (canceled)
 63. A fusion protein comprising a donor diversity scaffold domain inserted into a recipient diversity scaffold domain wherein the donor diversity scaffold domain comprises a donor scaffold and a donor interaction sequence and the recipient diversity scaffold domain comprises a recipient scaffold and a recipient interaction sequence.
 64. A binding member comprising a fusion protein according to claim 63 and a partner domain.
 65. A method of producing a binding member comprising: inserting a nucleic acid encoding a donor diversity scaffold domain into a nucleic acid encoding a recipient diversity scaffold domain to produce a chimeric nucleic acid encoding a fusion protein; wherein the donor diversity scaffold domain comprises a donor scaffold and a donor interaction sequence and the recipient diversity scaffold domain comprises a recipient scaffold and a recipient interaction sequence; expressing said chimeric nucleic acid to produce the fusion protein, and optionally, associating the fusion protein with a partner domain. 66-81. (canceled)
 82. The method according to claim 1, wherein one or both of the diversity scaffold domains comprise multiple disulphide bonds.
 83. The method according to claim 1, wherein the recipient diversity scaffold domain is selected from the group consisting of: an immunoglobulin, an immunoglobulin domain, a VH domain, a VL domain, a knottin, a Protein A, a “Designed ankyrin repeat protein” (DARPin), an adhiron, a fibronectin domain, an anticalin and a T7 phage gene 2 protein (Gp2).
 84. The method according to claim 83, wherein the recipient diversity scaffold domain is all or part of an immunoglobulin.
 85. The method according to claim 84, wherein the recipient diversity scaffold domain is all or part of an antibody variable domain.
 86. The method according to claim 85, wherein the recipient diversity scaffold domain is an antibody light chain variable (VL) domain.
 87. (canceled)
 88. The method according to claim 86, wherein the partner domain is an antibody heavy chain variable (VH) domain. 89-91. (canceled)
 92. The method according to claim 88, wherein the VL domain and the VH domain are connected by a flexible linker.
 93. The method according to claim 85, wherein the donor diversity scaffold domain replaces all or part of VH CDR1, VH CDR2, VH CDR3, VL CDR1, VL CDR2 or VL CDR3 of the antibody variable domain.
 94. The method according to claim 86, wherein the donor diversity scaffold domain replaces all or part of the CDR1 or CDR2 of the antibody VL domain. 95-97. (canceled)
 98. The method according to claim 1, wherein the donor diversity scaffold domain is selected from the group consisting of: an immunoglobulin, an immunoglobulin domain, a VH domain, a VL domain, a Protein A, cysteine-rich peptide, such as a venom peptide or knottin, a “Designed ankyrin repeat protein” (DARPin), an adhiron, a fibronectin domain, an anticalin and a T7 phage gene 2 protein (Gp2).
 99. The method according to claim 98, wherein the donor diversity scaffold domain is a cysteine-rich peptide.
 100. The method according to claim 99, wherein donor diversity scaffold domain is a venom peptide.
 101. The method according to claim 98, wherein the donor diversity scaffold domain is a knottin.
 102. The method according to claim 99, wherein the donor diversity scaffold domain is Ecballium elaterium trypsin inhibitor-II (EETI-II), MCoTI-II, Huwentoxin-IV, ProTx-II, SSm6a, Kaliotoxin, MoKa-1, Shk, PcTx1 and Conotoxin-ω, or a variant thereof.
 103. The method according to claim 98, wherein the native N and C terminals of the donor diversity scaffold domain are linked to the recipient diversity scaffold domain. 104-106. (canceled)
 107. The method according to claim 98, wherein artificial N and C terminals generated by cyclisation and linearization of the donor diversity scaffold domain are linked to the recipient diversity scaffold domain. 108-111. (canceled)
 112. A nucleic acid encoding a fusion protein according to claim
 63. 113-114. (canceled)
 115. A population of nucleic acids encoding a library according to claim
 54. 116-127. (canceled) 